Method of making nanoparticle colloid and nanoporous layer

ABSTRACT

This disclosure relates to a glucose-sensing electrode including a nanoporous metal layer and an electrolyte ion-blocking layer formed over the nanoporous metal layer. The nanoporous metal layer is capable of oxidizing both glucose and maltose without an enzyme specific to glucose in the glucose-sensing electrode. The electrolyte ion-blocking layer is configured to inhibit Na+, K+, Ca2+, Cl−, PO43− and CO32− from diffusing toward the nanoporous metal layer such that there is a substantial discontinuity of a combined concentration of Na+, K+, Ca2+, Cl−, PO43− and CO32− between over and below the electrolyte ion-blocking layer.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

Any and all applications for which a foreign or domestic priority claimis identified in the Application Data Sheet as filed with the presentapplication are hereby incorporated by reference under 37 CFR 1.57.

BACKGROUND Field

The present disclosure relates to glucose-sensing.

Discussion of the Related Technology

A high level of interest exists in the healthcare community and industryfor improving the technologies of sensing and monitoring blood glucoselevels. Today, most glucose sensors use an electrochemical method. Most,if not all, electrochemical sensors use enzyme-based electrochemicalsensors.

SUMMARY

One aspect of the invention provides a colloid composition comprising: anumber of clusters of nanoparticles dispersed in a liquid, wherein eachcluster comprises a number of nanoparticles that are clustered togetherto form a irregularly shaped body having a nano-sized or micro-sizedlength, wherein individual nanoparticles have a discrete body in agenerally oval or spherical shape with a diameter of about 2 nm to about5 nm, wherein interparticular gaps are formed between adjacentnanoparticles inside each cluster and have an interparticular gapdistance of about 0.5 nm to about 2 nm.

In the foregoing colloid composition, the interparticular gaps may bedistributed generally throughout in each cluster. The composition may besubstantially free of a surfactant. The liquid may comprise water,wherein the colloid composition may comprise a surfactant in an amountsmaller than 2 parts by weight with reference to 100 parts by weight ofthe nanoparticles contained therein. The nanoparticles contained in thecolloid composition may be in an amount between about 0.01 wt % andabout 2 wt % with reference to the total weight of the colloidcomposition. The nanoparticles contained in the colloid composition maybe in an amount between about 0.01 wt % and about 1 wt % with referenceto the total weight of the colloid composition.

Still in the foregoing colloid composition, the nanoparticles may beprimarily made of at least one selected from the group consisting ofplatinum (Pt), gold (Au), palladium (Pd), rhodium (Rh), titanium (Ti),ruthenium (Ru), tin (Sn), nickel (Ni), copper (Cu), indium (In),thallium (Tl), zirconium (Zr), iridium (Ir), and one or more oxides ofeach of the foregoing elements. The nanoparticles may be primarily madeof platinum (Pt), wherein the interparticular gaps may be distributedgenerally throughout in each cluster, wherein the colloid compositionmay comprise a surfactant in an amount smaller than 1 parts by weightwith reference to 100 parts by weight of the nanoparticles containedtherein, wherein the nanoparticles contained in the colloid compositionmay be in an amount between about 0.1 wt % and about 1 wt % withreference to the total weight of the colloid composition.

Another aspect of the invention provides a method of making a nanoporouslayer. The method comprises dispensing the foregoing colloid compositionover a substrate; subjecting the dispensed colloid composition to dryingsuch that the clusters contained in the dispensed composition aredeposited over the substrate and also stacked over one another toprovide a nanoporous layer over the substrate, wherein the nanoporouslayer comprises irregularly shaped bodies formed of the clusters thatare stacked over one another, wherein the irregularly shaped bodiescomprises a number of nanoparticles locally clustered together andinterparticular gaps formed between adjacent ones of the nanoparticlesin the irregularly shaped bodies, wherein the irregularly shaped bodiesare interconnected to provide a three-dimensional interconnected networkof irregularly shaped bodies, wherein irregularly shaped spaces areformed between adjacent portions of the irregularly shaped bodies andare nano-sized or micro-sized.

In the foregoing method, the nanoparticles may be generally in an ovalor spherical shape having a diameter of about 2 nm to about 5 nm. Theinterparticular gaps may have an interparticular gap distance of about0.5 nm to about 2 nm. The irregularly shaped spaces may beinterconnected to provide a three-dimensional interconnected network ofirregularly shaped spaces. The colloid composition may be dispensed in apredetermined amount to form the nanoporous layer having roughnessfactor between about 100 and about 2500. The nanoporous layer maycomprise a surfactant in an amount smaller than 0.5 parts by weight withreference to 100 parts by weight of the nanoparticles contained therein.

Another aspect of the invention provides a method of making a colloidcomposition. The method comprises: providing a liquid compositioncomprising a metal ion, a surfactant and a solvent, wherein thesurfactant is in a reverse micelle phase defining hydrophilic spaces;adding a reducing agent to the liquid composition to cause reduction ofthe metal ion, which forms a first colloid comprising metalnanoparticles and the surfactant, wherein in the first colloid the metalnanoparticles are dispersed along with the reverse micelle phase of thesurfactant; and removing the surfactant from the first colloid toprovide a second colloid comprising a number of clusters dispersed in aliquid, wherein each cluster comprises a number of nanoparticles thatare clustered together to form a irregularly shaped body having anano-sized or micro-sized length.

In the foregoing method of making, no electric potential may be appliedto the liquid composition for reduction of the metal ion therein. Thesurfactant may be a non-ionic surfactant capable of forming an isotropicreverse micelle phase. Individual nanoparticles may have a discrete bodyin a generally oval or spherical shape with a diameter of about 2 nm toabout 5 nm, wherein interparticular gaps may be formed between adjacentnanoparticles inside each cluster and have an interparticular gapdistance of about 0.5 nm to about 2 nm. Removing the surfactant removesa significant amount of the surfactant from the first colloid such thatthe second colloid is substantially free of the surfactant. Removing thesurfactant removes a significant amount of the surfactant from the firstcolloid such that the second colloid contains the surfactant in anamount smaller than 1 part by weight with reference to 100 parts byweight of the nanoparticles contained therein.

Still in the foregoing method of making, removing the surfactant maycomprise: centrifuging the first colloid; and collecting a bottomportion from a centrifuged composition. Removing the surfactant mayfurther comprise repeating a sequence of centrifuging and collectingmultiple times. Removing the surfactant may further comprise adding anacid or base to the first colloid prior to centrifuging. Removing thesurfactant may further comprise repeating a sequence of adding,centrifuging and collecting multiple times. The nanoparticles containedin the second colloid may be in an amount between about 10 wt % andabout 40 wt % with reference to the total weight of the composition. Thenanoparticles may be primarily made of at least one selected from thegroup consisting of platinum (Pt), gold (Au), palladium (Pd), rhodium(Rh), titanium (Ti), ruthenium (Ru), tin (Sn), nickel (Ni), copper (Cu),indium (In), thallium (Tl), zirconium (Zr), iridium (Ir), and one ormore oxides of each of the foregoing metals. The nanoparticles may beprimarily made of platinum (Pt), wherein the interparticular gaps may bedistributed generally throughout in each cluster, wherein thecomposition may comprise a surfactant in an amount smaller than 2 partsby weight with reference to 100 parts by weight of the nanoparticlescontained therein, wherein the nanoparticles contained in thecomposition may be in an amount of about 0.1 wt % and about 2 wt % withreference to the total weight of the composition.

Another aspect of the invention provides a method of making a nanoporouslayer. This method comprises the foregoing method of making a colloidcomposition to provide the second colloid; dispensing the second colloidover a substrate; subjecting the dispensed second colloid to drying suchthat the clusters contained in the dispensed composition are depositedover the substrate and also stacked over one another to provide ananoporous layer over the substrate, wherein the nanoporous layercomprises irregularly shaped bodies formed of the clusters that arestacked over one another, wherein the irregularly shaped bodiescomprises a number of nanoparticles locally clustered together andinterparticular gaps formed between adjacent ones of the nanoparticlesin the irregularly shaped bodies. The irregularly shaped bodies areinterconnected to provide a three-dimensional interconnected network ofirregularly shaped bodies, wherein irregularly shaped spaces are formedbetween adjacent portions of the irregularly shaped bodies and arenano-sized or micro-sized, wherein the irregularly shaped spaces areinterconnected to provide a three-dimensional interconnected network ofirregularly shaped spaces.

In the foregoing method of making a nanoporous layer, the nanoparticlesmay be generally in an oval or spherical shape having a diameter ofabout 2 nm to about 5 nm, wherein the interparticular gaps have aninterparticular gap distance of about 0.5 nm to about 2 nm. The colloidcomposition may be dispensed in a predetermined amount to form thenanoporous layer having roughness factor between about 100 and about2500. The nanoporous layer may comprise a surfactant smaller than 0.1parts by weight with reference to 100 parts by weight of thenanoparticles contained therein.

Another aspect of the invention provides a nanoporous structurecomprising: irregularly shaped bodies comprising a number ofnanoparticles locally clustered together and interparticular gaps formedbetween adjacent ones of the nanoparticles in the irregularly shapedbodies, wherein the nanoparticles may be generally in an oval orspherical shape having a diameter of about 2 nm to about 5 nm, whereinthe interparticular gaps have an interparticular gap distance of about0.5 nm to about 2 nm, wherein the irregularly shaped bodies may beinterconnected to provide a three-dimensional interconnected network ofirregularly shaped bodies, wherein irregularly shaped spaces are formedbetween adjacent portions of the irregularly shaped bodies and arenano-sized or micro-sized, wherein the irregularly shaped spaces areinterconnected to provide a three-dimensional interconnected network ofirregularly shaped spaces.

The foregoing nanoporous structure may be substantially free ofsurfactant molecules. In the foregoing nanoporous structure, theinterparticular gaps may be substantially free of nano-sized organicmolecules. The three-dimensional network of irregularly shaped bodiesand the three-dimensional network of irregularly shaped interclustergaps may be complementary to form the nanoporous structure. Theinterparticular gaps may be substantially interconnected themselves andmay be further connected to the three-dimensional interconnected networkof irregularly shaped intercluster gaps. The nanoporous structure may beformed by dispensing a solid-liquid colloid comprising irregularlyshaped discrete clusters dispersed in liquid and drying the dispensedsolid-liquid colloid, in which the irregularly shaped discrete clustersmay be stacked to provide the three-dimensional interconnected networkof irregularly shaped bodies and the three-dimensional interconnectednetwork of irregularly shaped intercluster gaps. The irregularly shapedintercluster gaps have a mean intercluster gap distance. Thenanoparticles may be made of at least one selected from the groupconsisting of platinum (Pt), gold (Au), palladium (Pd), rhodium (Rh),titanium (Ti), ruthenium (Ru), tin (Sn), nickel (Ni), copper (Cu),indium (In), thallium (Tl), zirconium (Zr), iridium (Ir), and one ormore oxides of each of the foregoing metals. The nanoporous structurehas roughness factor between about 100 and about 2500.

Another aspect of the invention provides a device comprising: asubstrate comprising a surface; and a nanoporous layer formed on thesurface and comprising the foregoing nanoporous structure. Still anotheraspect of the invention provides a non-enzymatic glucose-sensingelectrode comprising: at least one conductive layer comprising asurface; and a nanoporous layer formed on the surface and comprising theforegoing nanoporous structure, wherein the non-enzymaticglucose-sensing electrode does not comprise a glucose-specific enzyme.

In the foregoing device or electrode, the at least one conductive layermay comprise an electrically conductive metal layer and an electricallyconductive carbon layer formed on the electrically conductive metallayer. The device or electrode does not comprise a biocompatiblepolymeric material formed over the nanoporous layer. The device orelectrode may comprise a biocompatible polymeric material formed overthe nanoporous layer.

Still another aspect of the invention provides a one-time use glucosesensing device comprising: a reservoir configured to receive and hold atest liquid; and the foregoing electrode arranged with the reservoirsuch that the nanoporous layer may be to contact the test liquid whenthe test liquid may be held in the reservoir. In the one-time useglucose sensing device, the electrode does not comprise a biocompatiblepolymeric material formed over the nanoporous layer.

Still another aspect of the invention provides a continuous glucosemonitoring (CGM) device comprising: a hypodermic needle configured forcontacting interstitial fluid of a subject's body; and an electricalcircuit connected to the hypodermic needle, wherein the hypodermicneedle comprises the foregoing electrode and another electrode that areconnected to the electrical circuit.

A still another aspect of the invention provides a non-enzymaticglucose-sensing device comprising: a working electrode comprising asubstrate and a nanoporous layer formed over the substrate, the workingelectrode does not comprise a glucose-specific enzyme, wherein thenanoporous layer may comprise irregularly shaped bodies comprising anumber of nanoparticles locally clustered together, whereininterparticular gaps may be formed between adjacent ones of thenanoparticles in the irregularly shaped bodies, wherein thenanoparticles may be generally in an oval or spherical shape having adiameter of about 2 nm to about 5 nm, wherein the interparticular gapshave an interparticular gap distance of about 0.5 nm to about 2 nm,wherein the irregularly shaped bodies may be interconnected to provide athree-dimensional interconnected network of irregularly shaped bodiesextending generally throughout the nanoporous layer, wherein irregularlyshaped spaces may be formed between adjacent portions of the irregularlyshaped bodies and may be nano-sized or micro-sized, wherein theirregularly shaped spaces may be interconnected to provide athree-dimensional interconnected network of irregularly shaped spacesextending generally throughout the nanoporous layer, wherein thenanoporous layer may be configured to cause oxidation of glucosemolecule therein in the absence of a glucose-specific enzyme at a biasvoltage applied thereto between about 0.2 V and about 0.45 V.

In the foregoing non-enzymatic glucose-sensing device, the nanoporouslayer may be substantially free of surfactant molecules, wherein thesubstrate may comprise at least one conductive layer comprising anelectrically conductive or semiconductive material. The interparticulargaps may be substantially free of nano-sized organic molecules. Thethree-dimensional network of irregularly shaped bodies and thethree-dimensional network of irregularly shaped intercluster gaps may becomplementary to form the nanoporous layer. The interparticular gaps maybe substantially interconnected themselves and may be further connectedto the three-dimensional interconnected network of irregularly shapedintercluster gaps.

Still in the foregoing non-enzymatic glucose-sensing device, thenanoporous layer may be formed by dispensing a solid-liquid colloidcomprising irregularly shaped discrete clusters dispersed in liquid anddrying the dispensed solid-liquid colloid, in which the irregularlyshaped discrete clusters may be stacked to provide the three-dimensionalinterconnected network of irregularly shaped bodies and thethree-dimensional interconnected network of irregularly shapedintercluster gaps. The nanoparticles may be made of at least oneselected from the group consisting of platinum (Pt), gold (Au),palladium (Pd), rhodium (Rh), titanium (Ti), ruthenium (Ru), tin (Sn),nickel (Ni), copper (Cu), indium (In), thallium (Tl), zirconium (Zr),iridium (Ir), and one or more oxides of each of the foregoing metals.The nanoporous layer has roughness factor between about 100 and about2500. The nanoporous electrode may further comprise a maltose-blockinglayer formed over the nanoporous layer and configured to substantiallyblock maltose contained in the test fluid from passing therethroughwhile allowing glucose to pass therethrough. The maltose-blocking layermay comprise poly-phenylenediamine (poly-PD) in a morphology allowingglucose molecules to pass therethrough while effectively blockingmaltose molecules from passing therethrough. The bias voltage may be setto be in a range between 0.2 V and 0.45 V.

Still another aspect of the invention provides a non-enzymaticglucose-sensing system comprising: the foregoing non-enzymaticglucose-sensing device; a counter electrode; and a bias voltage supplyelectrically connected between the working electrode and the counterelectrode for supplying a bias voltage between the working electrode andcounter electrode.

Still another aspect of the invention provides a method of non-enzymaticglucose sensing. The method comprises: providing the foregoingnon-enzymatic glucose-sensing device; applying the bias voltage betweenthe working electrode and the counter electrode while a test fluidcontacts both the working electrode and the counter electrode, whichcauses oxidation of glucose contained in the test fluid at thenanoporous layer; measuring electric current from the working electrode;and processing the electric current with or without additional data toprovide a glucose level that corresponds to glucose contained in thetest fluid. The bias voltage may be set to be in a range between 0.2 Vand 0.45 V.

Another aspect of the invention provides a glucose-sensing electrode,which comprises: a substrate; a nanoporous metal layer formed over thesubstrate and capable of oxidizing both glucose and maltose without anenzyme specific to glucose or maltose in the glucose-sensing electrode;a maltose-blocking layer formed over the nanoporous metal layer. In theglucose-sensing electrode, the maltose-blocking layer has porosity thatpermits glucose to pass therethrough and inhibits maltose from passingtherethrough toward the nanoporous metal layer such that electriccurrent caused by oxidation of glucose alone in the nanoporous metallayer is higher than 10 nA/mMcm² and further such that electric currentcaused by oxidation of maltose alone in the nanoporous metal layer islower than 5 nA/mMcm² when a bias voltage of 0.2-0.45 V is applied tothe nanoporous metal layer relative to a reference electrode and whenthe maltose-blocking layer contacts liquid containing glucose in aconcentration of 4-20 mM and maltose in a concentration of 4-20 mM.

In the foregoing glucose-sensing electrode, the nanoporous metal layeris capable of oxidizing glucose such that electric current caused byoxidation of glucose alone is higher than 10 nA/mMcm² when applying abias voltage of 0.2-0.45 V and contacting liquid containing glucose in aconcentration of 4-20 mM without the maltose-blocking layer thereover.The nanoporous metal layer is further capable of oxidizing maltose suchthat electric current caused by oxidation of maltose alone in higherthan 10 nA/mMcm² when applying a bias voltage of 0.2-0.45 V and whencontacting liquid containing maltose in a concentration of 4-20 mMwithout the maltose-blocking layer thereover. The maltose-blocking layermay comprise poly-phenylenediamine (poly-PD) and have a thicknessbetween 10 nm and 40 nm. The maltose-blocking layer may consistessentially of poly-phenylenediamine (poly-PD) and have a thicknessbetween 10 nm and 35 nm. The maltose-blocking layer may consist ofpoly-phenylenediamine (poly-PD) and have a thickness between 10 nm and40 nm.

In the foregoing glucose-sensing electrode, the nanoporous metal layermay comprise irregularly shaped bodies comprising a number ofnanoparticles locally clustered together and interparticular gaps formedbetween adjacent ones of the nanoparticles in the irregularly shapedbodies. Here, the nanoparticles are generally in an oval or sphericalshape having a diameter of about 2 nm to about 5 nm. The interparticulargaps may have an interparticular gap distance of about 0.5 nm to about 2nm. The irregularly shaped bodies may be interconnected to provide athree-dimensional interconnected network of irregularly shaped bodies.Irregularly shaped spaces may be formed between adjacent portions of theirregularly shaped bodies and are nano-sized or micro-sized. Theirregularly shaped spaces may be interconnected to provide athree-dimensional interconnected network of irregularly shaped spaces.

The foregoing glucose-sensing electrode may further comprise anelectrolyte ion-blocking layer formed over the maltose-blocking layerand a biocompatibility layer formed over the electrolyte ion blockinglayer. The electrolyte ion-blocking layer is configured to inhibit Na⁺,K⁺, Ca²⁺, Cl⁻, PO₄ ³⁻ and CO₃ ²⁻ contained in the liquid from diffusingtoward the nanoporous metal layer such that there is a substantialdiscontinuity of a combined concentration of Na⁺, K⁺, Ca²⁺, Cl⁻, PO₄ ³⁻and CO₃ ²⁻ between over the electrolyte ion-blocking layer and below theelectrolyte ion-blocking layer. The electrolyte ion-blocking layer mayfacilitate conditioning of the glucose-sensing electrode such thatconditioning of the glucose-sensing electrode is complete within 30minutes from contacting the subject's bodily fluid with the applicationof the bias voltage of 0.2-0.45 V.

Another aspect of the invention provides an apparatus comprising: asingle integrated body comprising a subcutaneous portion and a terminalportion; the subcutaneous portion comprising the foregoingglucose-sensing electrode and the reference electrode, each of which isexposed for contacting interstitial fluid of a first subject when thesubcutaneous portion is subcutaneously inserted into the first subject'sbody; and the terminal portion configured for coupling with acounterpart device and comprising a first terminal electricallyconnected to the glucose-sensing electrode and a second terminalelectrically connected to the reference electrode.

Still another aspect of the invention provides an apparatus comprising:a single integrated body comprising the foregoing glucose-sensingelectrode and the reference electrode, the single integrated bodyfurther comprising a reservoir configured to at least temporarily hold atest fluid therein, wherein the glucose-sensing electrode and thereference electrode are arranged in the single integrated body such thatwhen the test fluid is held in the reservoir each of the glucose-sensingelectrode and the reference electrode is configured to contact the testfluid.

A further aspect of the invention provides a method of making aglucose-sensing electrode. The method comprises: providing a nanoporousmetal layer capable of oxidizing both glucose and maltose without anenzyme specific to glucose or maltose in the glucose-sensing electrode;forming a poly-phenylenediamine (poly-PD) film over the nanoporousplatinum layer such that the poly-PD film allows glucose to passtherethrough and blocks maltose from passing therethrough. Here, thepoly-PD film has porosity to permit glucose to pass therethrough and toinhibit maltose from passing therethrough toward the nanoporous metallayer such that electric current caused by oxidation of glucose alone inthe nanoporous metal layer is higher than 10 nA/mMcm² and further suchthat electric current caused by oxidation of maltose alone in thenanoporous metal layer is lower than 5 nA/mMcm², when a bias voltage of0.2-0.45 V is applied to the nanoporous metal layer relative to areference electrode and when the poly-PD film contacts liquid containingglucose in a concentration of 4-20 mM and maltose in a concentration of4-20 mM.

In the foregoing method of making glucose-sensing electrode, forming thepoly-PD film may comprise performing electrochemical polymerizationusing the nanoporous metal layer as an electrode for the electrochemicalpolymerization. Forming the poly-PD film may comprise providing apolymer layer comprising poly-PD and adjusting the porosity of thepolymer layer when the polymer layer may not have enough porosity topermit glucose to pass therethrough such that electric current caused byoxidation of glucose alone in the nanoporous metal layer is lower than10 nA/mMcm². Adjusting the porosity may comprise subjecting the polymerlayer to at least one electric shock while the polymer layer contacts anacidic solution. Forming the poly-PD film may comprise polymerizingpoly-PD from a liquid composition containing phenylenediamine at aconcentration, wherein when the concentration is higher than apredetermined value, forming the poly-PD film further comprisesadjusting the porosity of the polymer layer. Adjusting the porosity maycomprise subjecting the polymer layer to at least one electric shockwhile the polymer layer contacts an acidic solution.

In the foregoing method of making glucose-sensing electrode, forming thepoly-PD film may comprise providing a polymer layer comprising poly-PDwithout further adjusting the porosity of the polymer layer when thepolymer layer may have sufficient porosity to permit glucose to passtherethrough such that electric current caused by oxidation of glucosealone in the nanoporous metal layer is expected to be higher than 10nA/mMcm². Forming the poly-PD film may comprise polymerizing poly-PDfrom a liquid composition containing phenylenediamine at aconcentration, wherein when the concentration is lower than apredetermined value, the method does not comprise adjusting the porosityof the polymer layer to form the poly-PD film.

One aspect of the invention provides a glucose-sensing electrode, whichcomprises: an electrically conductive layer; a nanoporous metal layerformed over the electrically conductive layer; an electrolyteion-blocking layer formed over the nanoporous metal layer; and abiocompatibility layer formed over the electrolyte ion-blocking layer.The glucose-sensing electrode does not include a glucose-specificenzyme. When contacting liquid containing glucose, Na⁺, K⁺, Ca²⁺, Cl⁻,PO₄ ³⁻ and CO₃ ²⁻, the electrolyte ion-blocking layer is configured toinhibit Na⁺, K⁺, Ca²⁺, Cl⁻, PO₄ ³⁻ and CO₃ ²⁻ contained in the liquidfrom diffusing toward the nanoporous metal layer such that there is asubstantial discontinuity of a combined concentration of Na⁺, K⁺, Ca²⁺,Cl⁻, PO₄ ³⁻ and CO₃ ²⁻ between over the electrolyte ion-blocking layerand below the electrolyte ion-blocking layer.

In the foregoing glucose-sensing electrode, when applying a bias voltageof 0.2-0.45 V thereto relative to a reference electrode, theglucose-sensing electrode is configured to cause oxidation of glucose inthe nanoporous metal layer and configured to generate an electriccurrent that is a sum of a glucose-oxidation current caused by theglucose oxidation alone and a background current caused by otherelectrochemical interactions of the liquid and the glucose-sensingelectrode. When the liquid contains glucose at a concentration of 4-20mM (approximately 72-360 mg/dL), at steady state the glucose-oxidationcurrent is at a level higher than 10 nA/mMcm².

In the foregoing glucose-sensing electrode, the combined concentrationbelow the electrolyte ion-blocking layer is greater than 0% and lowerthan about 10% of the combined concentration above the electrolyteion-blocking layer. The combined concentration below the electrolyteion-blocking layer is greater than 0% and lower than about 5% of thecombined concentration above the electrolyte ion-blocking layer. Theelectrolyte ion-blocking layer may comprise a porous and hydrophobicpolymer layer that is configured to limit mobility of Na⁺, K⁺, Ca²⁺,Cl⁻, PO₄ ³⁻ and CO₃ ²⁻ therethrough while not limiting mobility ofglucose molecules therethrough.

In the foregoing glucose-sensing electrode, the electrolyte ion-blockinglayer may comprise at least one selected from the group consisting ofpoly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate)(PHEMA), and poly(methyl methacrylate-co-ethylene glycol dimethacrylate)(PMMA-EG-PMMA). The electrolyte ion-blocking layer may comprise at leastone selected from the group consisting of a copolymer ofmethylmethacrylate and butylmethacrylate, and polymers obtained frompolymerization of one or more monomers including branched or unbranchedC1-C8 alkylmethacrylate, branched or unbranched C1-C8cycloalkylmethacrylate, branched or unbranched C1-C8 alkylacrylate,branched or unbranched C1-C8 cycloalkylacrylate, and branched orunbranched C1-C8 cycloalkylmethacrylate, wherein the one or moremonomers are selected from the group consisting of methylmethacrylate,ethylmethacrylate, propylmethacrylate, butylmethacrylate,pentylmethacrylate, hexylmethacrylate, cyclohexylmethacrylate,2-ethylhexylmethacrylate, methylacrylate, ethylacrylate, propylacrylate,butylacrylate, pentylacrylate, hexylacrylate, cyclohexylacrylate, and2-ethylhexylacrylate.

In the foregoing glucose-sensing electrode, the glucose-sensingelectrode may be a continuous glucose monitoring (CGM) electrode,wherein the liquid is bodily fluid of a subject. The electrolyteion-blocking layer is configured to facilitate conditioning of theglucose-sensing electrode such that conditioning of the glucose-sensingelectrode is complete within 30 minutes from contacting the subject'sbodily fluid with the application of the bias voltage of 0.2-0.45 V.Conditioning of the glucose-sensing electrode may be considered ascomplete when a rate of decrease of the electric current is smaller thana first predetermined value and/or when the electric current stayssmaller than a second predetermined value.

The glucose-sensing electrode may further comprise a maltose-blockinglayer interposed between the nanoporous metal layer and the electrolyteion-blocking layer, wherein the maltose-blocking layer may comprisepoly-phenylenediamine (poly-PD). The maltose-blocking layer may beconfigured to let glucose pass therethrough and substantially blockmaltose from passing therethrough such that at steady state theglucose-oxidation current is at a level higher than 10 nA/mMcm² while amaltose oxidation current caused by oxidation of maltose alone is lowerthan 5 nA/mMcm².

The reference electrode may be configured to provide a reference levelof electric potential for the bias voltage applied to theglucose-sensing electrode, whether reduction of a chemical entity occursin the reference electrode or not. In a three-electrode electrochemicalcell, in addition to the reference electrode, a counter electrode isprovided for reduction of the chemical entity therein, whereas in atwo-electrode electrochemical cell, reduction of the chemical entityoccurs in the reference electrode.

In the foregoing glucose-sensing electrode, the nanoporous metal layermay comprise: irregularly shaped bodies comprising a number ofnanoparticles locally clustered together and interparticular gaps formedbetween adjacent ones of the nanoparticles in the irregularly shapedbodies, wherein the nanoparticles are generally in an oval or sphericalshape having a diameter of about 2 nm to about 5 nm, wherein theinterparticular gaps have an interparticular gap distance of about 0.5nm to about 2 nm. Here, the irregularly shaped bodies may beinterconnected to provide a three-dimensional interconnected network ofirregularly shaped bodies. Irregularly shaped spaces may be formedbetween adjacent portions of the irregularly shaped bodies and arenano-sized or micro-sized, and the irregularly shaped spaces areinterconnected to provide a three-dimensional interconnected network ofirregularly shaped spaces.

Another aspect of the invention provides a sensor apparatus comprising:a single integrated body comprising a subcutaneous portion and aterminal portion; the subcutaneous portion comprising a glucose-sensingelectrode and the reference electrode, each of which is exposed forcontacting interstitial fluid of a first subject when the subcutaneousportion is subcutaneously inserted into the first subject's body; andthe terminal portion configured for coupling with a counterpart deviceand comprising a first terminal electrically connected to theglucose-sensing electrode and a second terminal electrically connectedto the reference electrode. The glucose-sensing electrode may includeone or more features of the foregoing glucose-sensing electrode.

Another aspect of the invention provides a method of continuous glucosemonitoring. The method comprises: providing a sensor apparatus;subcutaneously inserting the subcutaneous portion of the glucose-sensingelectrode into a first subject's body such that the glucose-sensingelectrode and the reference electrode contact interstitial fluid of thefirst subject's body; causing to apply a bias voltage of 0.2-0.45 V tothe glucose-sensing electrode relative to the reference electrode;measuring electric current generated from the glucose-sensing electrode;computing a glucose level using an electric current value that isobtained by a measurement of the electric current within less than 1hour from later of subcutaneous insertion of the subcutaneous portionand application of the bias voltage; and presenting, on a display, thecomputed glucose level as that of the first subject within a rangebetween about 4 mM and about 20 mM (approximately between about 72 mg/dLand about 360 mg/dL). The glucose-sensing electrode may include one ormore features of the foregoing glucose-sensing electrode.

A further aspect of the invention provides a sensor apparatus,comprising: a substrate; a first electrode (or glucose-sensingelectrode) comprising a first electrically conductive layer formed overthe substrate and a glucose-oxidation layer formed over the firstelectrically conductive layer; a first terminal formed over thesubstrate and electrically connected to the first electrode; a secondelectrode comprising a second electrically conductive layer formed overthe substrate; a second terminal formed over the substrate andelectrically connected to the second electrode; a reference electrodecomprising a third electrically conductive layer formed over thesubstrate; and a third terminal formed over the substrate andelectrically connected to the reference electrode.

In the sensor apparatus, when the first electrode contacts liquidcontaining glucose and ascorbic acid and acetaminophen and when applyinga first bias voltage between the first and reference electrodes that issufficient to oxidize glucose in the glucose-oxidation layer, theglucose-oxidation layer of the first electrode is configured to causeoxidation of glucose and at least one of ascorbic acid and acetaminophentherein and further configured to generate a first electric currentcomprising a glucose component caused by the glucose oxidation and afirst interference component caused by oxidation of at least one ofascorbic acid and acetaminophen in the glucose-oxidation layer. Thesecond electrode is arranged in the apparatus such that, when the firstelectrode contacts the liquid, the second electrode also contacts thesame liquid. The second electrode does not comprise a layer configuredto cause oxidation of glucose therein such that, when applying a secondbias voltage between the second and reference electrodes, the secondelectrode is configured to cause oxidation of at least one of ascorbicacid and acetaminophen therein but not to cause oxidation of glucosetherein and further configured to generate a second electric currentcomprising a second interference component caused by oxidation of atleast one of ascorbic acid and acetaminophen in the second electrode andnot caused by oxidation of glucose. The apparatus is configured toprovide the first electric current at the first terminal and the secondelectric current at the second terminal.

The foregoing sensor apparatus may be configured to provide the secondelectric current in connection with the first electric current when itprovides the first electric current. The sensor apparatus may beconfigured to generate the first electric current and the secondelectric current at the same time. The sensor apparatus may beconfigured to provide the first electric current and the second electriccurrent along with information indicative of time of generating firstelectric current and the second electric current. The sensor apparatusmay be configured to provide the second electric current together withthe first electric current whenever it provides the first electriccurrent. In the foregoing sensor apparatus, the first electric currentmay further comprise a first background current caused by otherelectrochemical interactions of the liquid and the glucose-sensinglayer, wherein the second electric current may further comprise a secondbackground current caused by other electrochemical interactions of theliquid and the second electrode.

In the foregoing sensor apparatus, when the first bias voltage isbetween 0.2 V and 0.32 V, the glucose-oxidation layer is configured tooxidize glucose and ascorbic acid but not acetaminophen, and the firstinterference component is caused by oxidation of ascorbic acid and notby oxidation of acetaminophen. When the second bias voltage is between0.2 V and 0.32 V, the second electrode is configured to oxidize ascorbicacid but not acetaminophen and the second interference component iscaused by oxidation of ascorbic acid and not by oxidation ofacetaminophen. In the foregoing sensor apparatus, when the first biasvoltage is between 0.34 V and 0.45 V, the glucose-oxidation layer isconfigured to oxidize glucose, ascorbic acid and acetaminophen, and thefirst interference component is caused by oxidation of ascorbic acid andacetaminophen. When the second bias voltage is between 0.34 V and 0.45V, the second electrode is configured to oxidize ascorbic acid andacetaminophen and the second interference component is caused byoxidation of both ascorbic acid and acetaminophen.

In the foregoing sensor apparatus, the first electrode may furthercomprise a maltose-blocking layer comprising poly-phenylenediamine(poly-PD) formed on the glucose-oxidation layer. When contacting liquidcontaining glucose with a concentration of 4-20 mM (approximately 72-360mg/dL) and when applying the bias voltage, the maltose-blocking layer isconfigured to let glucose pass therethrough and substantially blockmaltose from passing therethrough such that at steady state theglucose-oxidation current is at a level higher than 10 nA/mMcm² while amaltose oxidation current caused by oxidation of maltose alone is lowerthan 5 nA/mMcm².

The foregoing sensor apparatus may be a continuous glucose monitoring(CGM) electrode module comprising a subcutaneous portion configured tosubcutaneously contact bodily fluid of a subject, wherein the first,second and reference electrodes are formed in the subcutaneous portion.In the foregoing sensor apparatus, the glucose-oxidation layer maycomprise a nanoporous metal layer, wherein the first electrode furthermay comprise: an electrolyte ion-blocking layer formed over thenanoporous metal layer and a biocompatibility layer formed over theelectrolyte ion-blocking layer. The electrolyte ion-blocking layer maybe configured to inhibit Na⁺, K⁺, Ca²⁺, Cl⁻, PO₄ ³⁻ and CO₃ ²⁻ containedin the liquid from diffusing toward the nanoporous metal layer such thatthere is a substantial discontinuity of a combined concentration of Na⁺,K⁺, Ca²⁺, Cl⁻, PO₄ ³⁻ and CO₃ ²⁻ between over the electrolyteion-blocking layer and below the electrolyte ion-blocking layer.

In the foregoing sensor apparatus, the electrolyte ion-blocking layermay comprise a porous and hydrophobic polymer layer that is configuredto limit mobility of Na⁺, K⁺, Ca²⁺, Cl⁻, PO₄ ³⁻ and CO₃ ²⁻ therethroughwhile not limiting mobility of glucose molecules therethrough, whereinthe electrolyte ion-blocking layer may comprise at least one selectedfrom the group consisting of poly(methyl methacrylate) (PMMA),poly(hydroxyethyl methacrylate) (PHEMA), and poly(methylmethacrylate-co-ethylene glycol dimethacrylate) (PMMA-EG-PMMA).

In the foregoing sensor apparatus, the electrolyte ion-blocking layermay be configured to facilitate conditioning of the glucose-sensingelectrode such that conditioning of the glucose-sensing electrode iscomplete within 30 minutes from contacting the subject's bodily fluidwith the application of the bias voltage of 0.2-0.45 V, in whichconditioning of the glucose-sensing electrode is considered as completeeither or both of when a rate of decrease of the electric current issmaller than a first predetermined value and when the electric currentstays smaller than a second predetermined value.

The foregoing sensor apparatus is a blood glucose monitoring (BGM)electrode module comprising a reservoir configured to receive blood,wherein when blood is received in the reservoir, the first, second andreference electrodes are configured to contact the blood. The first biasvoltage is between 0.2 V and 0.45 V, wherein the second bias voltage isthe same as or different from the first bias voltage. Theglucose-oxidation layer may comprise a nanoporous metal material or aglucose-specific enzyme configured to oxidize glucose. Theglucose-oxidation layer may comprise irregularly shaped bodiescomprising a number of nanoparticles locally clustered together andinterparticular gaps formed between adjacent ones of the nanoparticlesin the irregularly shaped bodies, wherein the nanoparticles aregenerally in an oval or spherical shape having a diameter of about 2 nmto about 5 nm, wherein the interparticular gaps have an interparticulargap distance of about 0.5 nm to about 2 nm. Here, the irregularly shapedbodies may be interconnected to provide a three-dimensionalinterconnected network of irregularly shaped bodies. Irregularly shapedspaces may be formed between adjacent portions of the irregularly shapedbodies and are nano-sized or micro-sized, and the irregularly shapedspaces are interconnected to provide a three-dimensional interconnectednetwork of irregularly shaped spaces.

Still another aspect of the invention provides a system comprising: theforegoing sensor apparatus that further comprises a terminal portion inwhich the first, second and third terminals are arranged; a counterpartapparatus comprising a first counterpart terminal, a second counterpartterminal, a third counterpart terminal, circuitry, and an electric powerconnected to the circuitry; and the counterpart apparatus furthercomprising a counterpart terminal portion configured to connect orengage with the terminal portion. Here, the first, second and thirdcounterpart terminals are arranged in the counterpart terminal portionsuch that, when the terminal portion of the sensor apparatus and thecounterpart terminal portion of the counterpart apparatus are connectedor engaged, the first terminal electrically connects to the firstcounterpart terminal, the second terminal electrically connects to thesecond counterpart terminal, and the third terminal electricallyconnects to the third counterpart terminal. The circuitry of thecounterpart apparatus is configured to provide the first bias voltagebetween the first counterpart terminal and the third counterpartterminal, and the circuitry of the counterpart apparatus is furtherconfigured to provide the second bias voltage between the secondcounterpart terminal and the third counterpart terminal.

In the foregoing system, the counterpart apparatus may comprise awireless communication module configured to wirelessly communicate witha wirelessly paired computing device that comprises at least oneprocessor and at least one memory. The counterpart apparatus may beconfigured to receive the first electric current at the firstcounterpart terminal and the second electric current at the secondcounterpart terminal. The counterpart apparatus may be configured totransmit the second electric current together or in connection with thefirst electric current when it transmits the first electric current. Thefirst electric current may be transmitted with a first time stamp, andthe second electric current may be transmitted with a second time stamp,wherein the first and second time stamps indicate an identical time.

The foregoing system may further comprise software installed andexecutable by the at least one processor of the wirelessly pairedcomputing device. Upon execution, the software is configured to performa method comprising: causing to store, in the at least one memory of thecomputing device, the first electric current and the second electriccurrent received together or in connection with each other from thecounterpart apparatus; processing the first electric current and thesecond electric current to provide a value indicative of the oxidationof glucose in the glucose-oxidation layer of the first electrode of thesensor apparatus; and causing to present the value or its correspondinginformation on a display of the computing device.

In the foregoing system, either or both of the first electric currentand the second electric current may be in the form of continuoussignals, wherein processing the first electric current and the secondelectric current may comprise processing values of the first electriccurrent and the second electric current obtained at the same time. Here,processing values may comprise subtracting the second electric currentfrom the first electric current. The first electric current and thesecond electric current may be stored in connection with each other inthe at least one memory. The foregoing system may further comprisesoftware installed and executable in the wirelessly paired computingdevice. Upon execution the software is configured to perform dataprocessing to obtain a level of glucose contained in the liquid that thefirst electrode of the sensor apparatus contacts using the firstelectric current and the second electric current received from thecounterpart apparatus. Here, the software requires the second electriccurrent when processing to obtain the level of glucose.

In the foregoing system, the counterpart apparatus may further compriseat least one processor, at least one memory, and software stored in theat least one memory and executable by the at least one processor. Uponexecution the software is configured to perform a method comprising:causing to store, in the at least one memory, the first electric currentand the second electric current received together or in connection witheach other from the sensor apparatus; and processing the first electriccurrent and the second electric current to provide a value indicative ofthe oxidation of glucose in the glucose-oxidation layer of the firstelectrode of the sensor apparatus. Here, processing may comprisesubtracting the second electric current from the first electric current.Either or both of the first electric current and the second electriccurrent may be in the form of continuous signals, wherein processing thefirst electric current and the second electric current may compriseprocessing values of the first electric current and the second electriccurrent obtained at the same time. The counterpart device may furthercomprise a display, wherein the method further may comprise causing topresent the value or its corresponding information on the display. Thecounterpart device may further comprise a wireless communication moduleconfigured to wirelessly pair with a device that comprises a display,wherein the method may further comprise causing to transmit data to thewirelessly paired device for presenting the value or its correspondinginformation on the display of the wirelessly paired device.

Still another aspect of the invention provides a method ofelectrochemical sensing. The method comprises: providing a sensorapparatus comprising a first electrode that comprises aglucose-oxidation layer capable of oxidizing glucose, a second electrodethat does not comprise a layer capable of oxidizing glucose, and areference electrode; causing the first, second and reference electrodesto contact liquid containing glucose and ascorbic acid andacetaminophen; causing to apply a first bias voltage between the firstand reference electrode that is sufficient to oxidize glucose in theglucose-oxidation layer such that glucose and at least one of ascorbicacid and acetaminophen are oxidized in the glucose-oxidation layer andfurther such that a first electric current is generated from the firstelectrode, wherein the first electric current comprises a glucosecomponent caused by the glucose oxidation and a first interferencecomponent caused by oxidation of at least one of ascorbic acid andacetaminophen; causing to apply a second bias voltage between the secondand reference electrodes such that at least one of ascorbic acid andacetaminophen is oxidized in the second electrode but glucose is notoxidized therein and further such that a second electric current isgenerated from the second electrode, wherein the second electric currentcomprises a second interference component caused by oxidation of atleast one of ascorbic acid and acetaminophen in the second electrode;and providing the first electric current and the second electric currentfor processing, wherein when the first electric current is provided forprocessing, the second electric current also is provided in connectionwith the first electric current.

In the foregoing method, the first electric current and the secondelectric current may be generated at the same time or one after anotherwithin a reasonable period of time in which the glucose level does notchange substantially or more than a predetermined tolerance level. Thefirst electric current may be provided along with information indicativeof time of generating the first electric current, wherein the secondelectric current may be provided along with information indicative oftime of generating the second electric current. The second electriccurrent may be provided together with the first electric currentwhenever the first electric current is provided. In the foregoingmethod, the first bias voltage is applied between 0.2 V and 0.32 V tocause the glucose-oxidation layer to oxidize glucoses and ascorbic acidbut to not oxidize acetaminophen, in which the first interferencecomponent is caused by oxidation of ascorbic acid and not by oxidationof acetaminophen; the second bias voltage is applied between 0.2 V and0.32 V to cause the second electrode to oxidize ascorbic acid but to notoxidize acetaminophen, in which the second interference component iscaused by oxidation of ascorbic acid and not by oxidation ofacetaminophen. In the alternative, the first bias voltage is appliedbetween 0.34 V and 0.45 V to cause the glucose-oxidation layer tooxidize glucose, ascorbic acid and acetaminophen, in which the firstinterference component is caused by oxidation of ascorbic acid andacetaminophen; the second bias voltage is applied between 0.34 V and0.45 V to cause the second electrode to oxidize ascorbic acid andacetaminophen, in which the second interference component is caused byoxidation of both ascorbic acid and acetaminophen.

In the foregoing method, the sensor apparatus may further comprise amaltose-blocking layer formed over the glucose-oxidation layer andcomprising poly-phenylenediamine (poly-PD). The sensor apparatus may bea continuous glucose monitoring (CGM) electrode module comprising asubcutaneous portion configured to subcutaneously contact bodily fluidof a subject, wherein the first, second and reference electrodes areformed in the subcutaneous portion, wherein causing the first, secondand reference electrodes to contact liquid may comprise subcutaneouslyinserting the subcutaneous portion into a subject's body. Theglucose-oxidation layer may comprise a nanoporous metal layer, whereinthe first electrode further may comprise: an electrolyte ion-blockinglayer formed over the nanoporous metal layer and a biocompatibilitylayer formed over the electrolyte ion-blocking layer. The electrolyteion-blocking layer inhibits Na⁺, K⁺, Ca²⁺, Cl⁻, PO₄ ³⁻ and CO₃ ²⁻contained in the liquid from diffusing toward the nanoporous metal layersuch that there is a substantial discontinuity of a combinedconcentration of Na⁺, K⁺, Ca²⁺, Cl⁻, PO₄ ³⁻ and CO₃ ²⁻ between over theelectrolyte ion-blocking layer and below the electrolyte ion-blockinglayer.

In the foregoing method, the sensor apparatus is a blood glucosemonitoring (BGM) electrode module comprising a reservoir, whereincausing the first, second and reference electrodes to contact liquid maycomprise providing a blood sample in the reservoir. Theglucose-oxidation layer may comprise irregularly shaped bodiescomprising a number of nanoparticles locally clustered together andinterparticular gaps formed between adjacent ones of the nanoparticlesin the irregularly shaped bodies, wherein the nanoparticles aregenerally in an oval or spherical shape having a diameter of about 2 nmto about 5 nm, wherein the interparticular gaps have an interparticulargap distance of about 0.5 nm to about 2 nm. The irregularly shapedbodies may be interconnected to provide a three-dimensionalinterconnected network of irregularly shaped bodies. Irregularly shapedspaces may be formed between adjacent portions of the irregularly shapedbodies and are nano-sized or micro-sized, and the irregularly shapedspaces may be interconnected to provide a three-dimensionalinterconnected network of irregularly shaped spaces.

In the foregoing method, the sensor apparatus may further comprise afirst terminal electrically connected to the first electrode, a secondterminal electrically connected to the second electrode, and a thirdterminal electrically connected to the reference electrode. The sensorapparatus may further comprise a terminal portion in which the first,second and third terminals are arranged, wherein causing to apply thefirst bias voltage and the second bias voltage may comprise connecting acounterpart device that comprises a first counterpart terminal, a secondcounterpart terminal, a third counterpart terminal, circuitry, and anelectric power connected to the circuitry. The counterpart apparatus mayfurther comprise a counterpart terminal portion for connecting orengaging with the terminal portion of the sensor apparatus. The first,second and third counterpart terminals may be arranged in thecounterpart terminal portion such that, when the terminal portion of thesensor apparatus and the counterpart terminal portion of the counterpartapparatus are connected or engaged, the first terminal electricallyconnects to the first counterpart terminal, the second terminalelectrically connects to the second counterpart terminal, and the thirdterminal electrically connects to the third counterpart terminal. Thecircuitry of the counterpart apparatus may provide the first biasvoltage between the first counterpart terminal and the third counterpartterminal; the circuitry of the counterpart apparatus may provide thesecond bias voltage between the second counterpart terminal and thethird counterpart terminal.

Still another aspect of the invention provides a method of providing ordetermining a glucose level. The method comprises: providing softwarestored in at least one memory and executable by at least one processorprovided in the sensor apparatus or another device; executing, with theat least one processor, the software to process the first electriccurrent and the second electric current to provide a value indicative ofthe oxidation of glucose in the glucose-oxidation layer of the firstelectrode of the sensor apparatus; and causing to present the value orits corresponding information on a display provided in the sensorapparatus, the other device or still another device.

In the foregoing method, the at least one memory and the at least oneprocessor are provided in the other device. The method further maycomprise: transmitting the first electric current and the secondelectric current to the other device; and prior to executing, causing tostore, in the at least one memory, the first electric current and thesecond electric current received together or in connection with eachother. In the foregoing method, the first electric current istransmitted with a first time stamp, and the second electric current istransmitted with a second time stamp, wherein the first and second timestamps indicate an identical time. In the foregoing method, either orboth of the first electric current and the second electric current maybe in the form of continuous signals, wherein processing the firstelectric current and the second electric current may comprise processingvalues of the first electric current and the second electric currentobtained at the same time. In the foregoing method, processing maycomprise subtracting the second electric current from the first electriccurrent.

Still another aspect of the invention provides a sensor apparatuscomprising: a working electrode comprising a nanoporous metal layer; anda reference electrode; and a bias voltage applied between the workingelectrode and the reference electrode, wherein no glucose-specificenzyme is present in the working electrode.

In the sensor apparatus, the nanoporous metal layer comprisesirregularly shaped bodies comprising a number of nanoparticles locallyclustered together and interparticular gaps formed between adjacent onesof the nanoparticles in the irregularly shaped bodies, and thenanoparticles are generally in an oval or spherical shape having adiameter of about 2 nm to about 5 nm, wherein the interparticular gapshave an interparticular gap distance of about 0.5 nm to about 2 nm. Theirregularly shaped bodies may be interconnected to provide athree-dimensional interconnected network of irregularly shaped bodies.Irregularly shaped spaces are formed between adjacent portions of theirregularly shaped bodies and are nano-sized or micro-sized, and theirregularly shaped spaces are interconnected to provide athree-dimensional interconnected network of irregularly shaped spaces.In the sensor apparatus, the bias voltage is set to be sufficient tocause oxidation of glucose at the nanoporous metal layer but notsufficient to cause oxidation of acetaminophen at the nanoporous metallayer, wherein the bias voltage is set within a range between about 0.20V and about 0.32 V.

The sensor apparatus may comprise a continuous glucose monitoring (CGM)electrode module comprising a subcutaneous portion configured tosubcutaneously contact bodily fluid of a subject, wherein the workingelectrode and the reference electrode are formed in the subcutaneousportion. The working electrode may further comprise: an electrolyteion-blocking layer formed over the nanoporous metal layer; and abiocompatibility layer formed over the electrolyte ion-blocking layer.The electrolyte ion-blocking layer may be configured to inhibit Na⁺, K⁺,Ca²⁺, Cl⁻, PO₄ ³⁻ and CO₃ ² contained in the liquid from diffusingtoward the nanoporous metal layer such that there is a substantialdiscontinuity of a combined concentration of Na⁺, K⁺, Ca²⁺, Cl⁻, PO₄ ³⁻and CO₃ ² between over the electrolyte ion-blocking layer and below theelectrolyte ion-blocking layer. The electrolyte ion-blocking layer maybe configured to facilitate conditioning of the working electrode suchthat conditioning of the working electrode is complete within 30 minutesfrom contacting the subject's bodily fluid with the application of thebias voltage.

The foregoing sensor apparatus may further comprise: a maltose-blockinglayer comprising poly-phenylenediamine (poly-PD) and interposed betweenthe nanoporous metal layer and the electrolyte ion-blocking layer. Whencontacting liquid containing maltose and glucose with a concentration of4-20 mM (approximately 72-360 mg/dL) and when applying the bias voltage,the maltose-blocking layer is configured to let glucose passtherethrough and substantially block maltose from passing therethroughsuch that at steady state the glucose-oxidation current is at a levelhigher than 10 nA/mMcm² while a maltose oxidation current caused byoxidation of maltose alone is lower than 5 nA/mMcm².

Still another aspect of the invention provides a method of glucosesensing. The method comprises: providing one of the foregoing sensorapparatus; and applying a bias voltage between the working electrode (orglucose-sensing electrode) and the reference electrode within a rangebetween about 0.20 V and about 0.32 V. Here, application of the biasvoltage causes oxidation of glucose in the nanoporous metal layer suchthat a glucose-oxidation current caused by glucose oxidation alone is ata level higher than 10 nA/mMcm², whereas application of the bias voltagedoes not cause sufficient oxidation of acetaminophen in the nanoporousmetal layer such that an acetaminophen oxidation current caused byacetaminophen oxidation in the nanoporous metal layer is lower than 5nA/mMcm².

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor.

FIG. 1 illustrates a conceptual electrochemical glucose-sensing systemaccording to embodiments of the invention.

FIG. 2 illustrates a working electrode for an enzymatic glucose-sensingsystem according to an embodiment.

FIG. 3 illustrates a working electrode including a nanoporous layer fora non-enzymatic sensing system according to an embodiment.

FIG. 4 illustrates a nanoporous layer's top surface and depth.

FIG. 5A illustrates a clustered morphology of a nanoporous layeraccording to an embodiment.

FIG. 5B is a TEM photographic image of clusters according to anembodiment.

FIG. 5C is a zoomed-in image of the TEM photographic image of FIG. 5B.

FIG. 5D is an SEM photographic image of a nanoporous layer taken fromits top according to an embodiment.

FIG. 6A is a flowchart for making a clustered nanoporous layer accordingto an embodiment.

FIG. 6B is a flowchart for making a clustered nanoporous layer accordingto another embodiment.

FIG. 7 is an example phase diagram of a surfactant showing differentphases.

FIG. 8 illustrates a reverse micelle phase and a nanoparticle-surfactantcolloid according to an embodiment.

FIG. 9 includes TEM photographic images of nanoparticle clustersaccording to an embodiment.

FIG. 10A illustrates a non-clustered morphology of a nanoporous layeraccording to an embodiment.

FIG. 10B is a TEM photographic image of a non-clustered morphology of ananoporous layer formed on a metal surface according to an embodiment.

FIG. 11 is a flowchart for making a non-clustered nanoporous layeraccording to an embodiment.

FIG. 12 is a flowchart for making a hexagonal nanostructure according toan embodiment.

FIG. 13A illustrates formation of a hexagonal arrangement according toan embodiment.

FIG. 13B illustrates deposition of metal using a hexagonal arrangementof liquid crystalline phase.

FIG. 14 shows a particle size distribution for nanoparticle-surfactantcolloid prepared according to an embodiment.

FIG. 15 shows a particle size distribution for cluster colloid preparedaccording to an embodiment.

FIGS. 16A and 16B illustrate a cross-section of an electrode base and anon-enzymatic glucose-sensing working electrode, respectively, accordingto embodiments.

FIGS. 17A-17C are SEM photographs of glucose-sensing working electrodesaccording to embodiments.

FIG. 18 is a profile of electric current generated by oxidation ofglucose and other materials in PBS according to embodiments.

FIG. 19 is a profile of electric current generated by oxidation ofglucose and other materials in human serum according to embodiments.

FIG. 20 is a structural formula of a maltose molecule.

FIG. 21 illustrates a non-enzymatic working electrode including amaltose-blocking layer according to an embodiment.

FIG. 22 illustrates scanning of oxidation voltage during cyclicvoltammetric electrochemical polymerization of phenylenediamineaccording to embodiment.

FIG. 23 illustrates a chronoamperometry setup for an electric shocktreatment to adjust porosity of a porous polymer layer according to anembodiment.

FIG. 24 is a flowchart for making a maltose-blocking layer according toan embodiment.

FIGS. 25-30 show electric currents monitored using glucose-sensingelectrode with a maltose-blocking layer according to embodiments, inwhich the electric current signals are presented in color as they wouldnot be easily seen in black and white.

FIG. 31 illustrates a CGM working electrode according to an embodiment.

FIG. 32 illustrates electrolyte concentration drop across the thicknessof an electrolyte ion-blocking layer according to an embodiment.

FIG. 33 illustrates a CGM electrode unit according to an embodiment.

FIG. 34 is a flowchart for fabricating a CGM electrode unit according toan embodiment.

FIGS. 35-37 illustrate top and cross-sectional views of intermediateproducts at various stages of fabricating the CGM electrode of FIG. 33,in which each cross-section is taken along the line 3501 and viewed inthe arrow direction.

FIGS. 38A and 38B illustrate a cross-section of an intermediate productafter forming nanoporous layer and a CGM working electrode withfunctional layers, respectively, according to embodiments.

FIG. 39 illustrates a disposable glucose-sensing cartridge according toembodiments.

FIG. 40 illustrates a two-electrode glucose-sensing system according toan embodiment.

FIG. 41 illustrates a CGM electrode unit for a two-electrodeglucose-sensing system according to an embodiment.

FIG. 42A is a profile of electric current generated by oxidation ofglucose according to an embodiment in which the working electrode doesnot include an electrolyte ion-blocking layer. FIG. 42B is an enlargedview of a portion of the profile of FIG. 42A.

FIG. 43 is a profile of electric current generated by oxidation ofglucose according to an embodiment in which the working electrodeincludes an electrolyte ion-blocking layer.

FIG. 44 is a comparison of time for conditioning working electrodes withand without an electrolyte ion-blocking layer.

FIGS. 45A, 45B and 45C are photographs of a potentiostat according to anembodiment.

FIG. 46 is a graph showing CGM monitoring of a rat's glucose level usinga non-enzymatic CGM electrode module according to an embodiment.

FIG. 47 is Clarke Error Grid for the non-enzymatic CGM electrode moduleaccording to an embodiment.

DETAILED DESCRIPTION OF EMBODIMENTS

The presently disclosed subject matter now will be described anddiscussed in more detail in terms of some specific embodiments andexamples with reference to the accompanying drawings, in which some, butnot all embodiments of the invention are shown. Like numbers refer tolike elements or parts throughout. The presently disclosed subjectmatter may be embodied in many different forms and should not beconstrued as limited to the specific embodiments set forth herein.Rather, these embodiments are provided so that this disclosure willsatisfy applicable legal requirements. Indeed, many modifications andother embodiments of the presently disclosed subject matter will come tothe mind of one skilled in the art to which the presently disclosedsubject matter pertains. Therefore, it is to be understood that thepresently disclosed subject matter is not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.

Electrochemical Glucose-Sensing System Electrochemical Glucose Detection

Electrochemical glucose sensing measures glucose concentration in anelectrolyte solution. FIG. 1 conceptually illustrates an electrochemicalglucose-sensing system 101 for detecting a glucose concentration in atest fluid or electrolyte solution102. The system 101 includes a workingor sensing electrode 103, a counter electrode 105 and a referenceelectrode 106 that are connected to a potentiostat 104 and in contactwith the test fluid 102. In embodiments, the potentiostat includeselectric circuitry for functioning as a voltage source 109 and a currentsensor 108. The voltage source 109 provides a bias voltage that drivesredox reactions at the working electrode 103 and counter electrode 105.The potentiostat further includes an electric circuitry such as anop-amp 107 for maintaining the bias voltage at the working electrode 103relative to the reference electrode 106. The current sensor 108 detectselectric current generated by redox reactions involving glucosecontained in the test fluid 102.

Enzymatic Glucose-Sensing Electrode

Most, if not all, electrochemical glucose-sensing systems utilize aglucose-specific enzyme for the detection of glucose molecules. FIG. 2illustrates a working electrode 103E for an enzymatic glucose-sensingsystem, i.e., an enzymatic glucose-sensing electrode. The terms“glucose-sensing electrode” and “working electrode” are interchangeablyused in the present disclosure. The enzymatic working electrode 103Eincludes a conductive layer 110 and an enzyme layer 111. Optionally, theenzymatic working electrode 103E may include at least one functionallayer 112 over the enzyme layer 111 as in FIG. 2. Alternatively,although not illustrated, at least one functional layer may be locatedbetween the enzyme layer 111 and conductive layer 110. The enzyme layer111 contains glucose-specific enzyme molecules 115, which are kepttherein by an immobilizer 113. When glucose molecules contact theglucose-specific enzyme, the enzyme catalyzes oxidation of glucose togluconolactone. Electrons from glucose oxidation are ultimatelytransferred to the conductive layer 110 for generating electric currentin the electrical circuit of the electrochemical sensing system 101.

Glucose Oxidase

In some enzymatic glucose-sensing systems, the enzymatic workingelectrode 103E includes glucose oxidase (GOx). Glucose oxidase 115transfers electrons to molecular oxygen staying near the enzyme, and themolecular oxygen is reduced to hydrogen peroxide. With a proper biasvoltage applied in the system, the conductive layer 110 oxidizeshydrogen peroxide and takes electrons therefrom, which generateselectric current indicative of the glucose concentration in the testfluid 102.

Glucose Dehydrogenase

In other enzymatic glucose-sensing systems, the enzymatic workingelectrode 103E includes glucose dehydrogenase (GDH). Unlike glucoseoxidase, glucose dehydrogenase does not use oxygen and instead transferselectrons to other adjacent chemical entities referred to as electronmediator, which then transfers electrons from the glucose oxidation tothe conductive layer 110. The electron mediator may be contained in theenzyme layer 111. Alternatively, the electron mediator may be providedin a separate layer (not shown) between the enzyme layer 111 and theconductive layer 110. While glucose dehydrogenase has some advantage ofsensitivity over glucose oxidase, this enzyme oxidizes maltose as wellas glucose, which interferes with accurate sensing of the glucoseconcentration.

Non-enzymatic Glucose-Sensing Electrode

Non-enzymatic electrochemical glucose-sensing systems do not use aglucose-specific enzyme or any enzyme for the detection glucose.Instead, non-enzymatic glucose-sensing systems have a non-enzymaticworking electrode that detects glucose without a glucose-specificenzyme. In embodiments, the non-enzymatic working electrode includes atleast one glucose oxidation layer that enables oxidation of glucosemolecules at a moderate level of bias voltage. Generally, the higher thebias voltage, the more likely glucose oxidation occurs at the at leastone glucose oxidation layer. However, because other chemical entitieswill also be oxidized at a high bias voltage, there is a limit for thebias voltage. Thus, non-enzymatic electrochemical glucose sensing relieson a material that oxidizes glucose at a bias voltage that does notcause oxidation of other chemical entities contained in the test fluid.

Nanoporous Layer for Non-enzymatic Glucose-Sensing Electrode

FIG. 3 illustrates a non-enzymatic working electrode (simply “workingelectrode”) 103NE that includes an electrically conductive layer 110 anda nanoporous glucose oxidation layer (or nanoporous layer) 117. Inembodiments, the nanoporous layer 117 includes nanoporous internalstructures for causing, enabling or facilitating oxidation of glucose ata moderate bias voltage. When glucose oxidation occurs, the conductivelayer 110 takes electrons from glucose oxidation and electrical currentis generated in the electrical circuit. The electrical current can bedetected by the current sensor 108 and interpreted by hardware andsoftware of the system. Optionally, the working electrode 103NE mayinclude at least one functional layer 112 over the nanoporous layer 117or between the nanoporous layer 117 and conductive layer 110 (notshown).

Conductive Layer—Materials

With the bias voltage, the conductive layer 110 of FIGS. 2 and 3 takeselectrons from glucose oxidation and transfers them to the currentsensor 108. In embodiments, the conductive layer 110 includes or is madeof at least one electrically conductive material and is connected toelectrical circuit of the system 101. In some embodiments, given thesmall scale of the conductive layer 110, semiconductive materials may beused instead of electrically conductive material. Non-limiting examplesfor a material of the conductive layer includes platinum (Pt), gold(Au), silver (Ag), ruthenium (Ru), stainless steel, silicon (amorphous,poly and single crystalline), conductive carbon materials, includinggraphite, graphene, fluorene, carbon nanotubes. In the embodiments, theconductive layer 110 does not include nanoporous internal structures ofthe glucose oxidation layer 117.

Conductive Layer—Configurations

In embodiments, the conductive layer 110 may be formed of a single layerof a homogeneous material. In the alternative, the conductive layer 110may include multiple sublayers made of different materials. In someembodiments, the conductive layer 110 includes top sublayer and one ormore sublayers under the top sublayer. In embodiments, the top sublayerdoes not contain silver, copper, aluminum or other conductive materialsthat are prone to oxidation more than silver, copper or aluminum. Thetop sublayer may be less electrically conductive than the othersublayer(s). In some embodiments, the conductive layer 110 includes aconductive carbon layer as the top sublayer and a silver layer asanother sublayer under the carbon layer. The conductive layer 110 has athickness that can vary significantly depending upon particularexamples. In some embodiments, the conductive layer 110 may be omitted,and the nanoporous layer is directly connected to the current sensor viaan electrically conductive wire or connection.

Counter Electrode

With the bias voltage, reduction of a chemical entity occurs at thecounter electrode 105. In embodiments, the counter electrode 105includes at least one electrically conductive or semiconductive materialand is connected to electrical circuit of the system 101. Inembodiments, the counter electrode 105 may be formed of a single layerof a homogenous material or multiple layers made of different materials.The conductive or semiconductive materials for the conductive layer 110may also be used in the counter electrode 105 although not the samematerials are used in the conductive layer 110 and in the counterelectrode 105 in a particular system.

Reference Electrode

The reference electrode 106 provides stability in the electrochemicalsensing system by maintaining the bias voltage between the sensingelectrode 103 and the reference electrode. As a result, glucoseoxidation can continue at the sensing electrode 103 even if reduction atthe counter electrode 105 is not at the same rate as the oxidation atthe sensing electrode 103. In some embodiments, the counter electrode105 may be omitted, and the reference electrode 106 may serve dualfunctions of the counter and reference electrodes. In embodiments, thereference electrode 106 may be formed of a single layer of a homogenousmaterial or multiple layers made of different materials. The conductiveor semiconductive materials for the conductive layer 110 may also beused in the reference electrode 105 although not the same materials areused in the conductive layer 110 and in the reference electrode 106 in aparticular system. In some embodiments, the reference electrode 106 mayinclude a salt layer over the conductive or semiconductive materiallayer. For example, the salt layer is made of or includes silverchloride (AgCl).

Current Sensor

The current sensor 108 measures electric current flowing from theworking electrode 103. The current sensor 108 may amperometricallydetect electric current flowing at a specific point in time. In thealternative, the current sensor 108 may be a coulometriccharge-measuring device.

Test Fluid

In embodiments, the test fluid is a biological fluid of human or animal,although not limited thereto. In some embodiments, the test fluid is aliquid mixture including a biological fluid and at least one additionalsubstance added to the biological fluid. The biological fluid includes,for example, blood, interstitial fluid, cerebral spinal fluid, lymphfluid or urine, although not limited thereto. In some embodiments, thetest fluid includes a non-biological liquid prepared for experiments.

Bias Voltage

The bias voltage applied between the working electrode 103NE andreference electrode 106 is at or about 0.10, 0.11, 0.12, 0.13, 0.14,0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26,0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38,0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45 or 0.46 V. In embodiments, thebias voltage applied may be within a range formed by selecting any twonumbers (two voltage values) listed in the immediately previoussentence, e.g., between about 0.20 V and about 0.30 V, between about0.30 V and about 0.40 V, between about 0.28 V and about 0.40 V, betweenabout 0.30 V and about 0.38 V, between about 0.28 V and about 0.36 V,etc.

Nanoporous Layer Nanoporous Layer

The nanoporous layer 117 for the working electrode 103NE includesnano-size internal structures such as cavities, spaces and openings(collectively “nano-pores” or “nanopores”). In embodiments, nanopores ofthe nanoporous layer 117 enable or facilitate oxidation of glucose, andglucose concentration can be measured based on electric current causedby glucose oxidation. Although any aspects of the invention are notbound by any theory or belief, it is conceivable that glucose oxidationoccurs when glucose molecules enter nanopores and contact internalsurfaces more often and for a longer time in the nanoporous layer 117than on a non-porous surface of an electrode.

No Enzyme and No Electron Mediator

With the incorporation of the nanoporous layer 117, the workingelectrode 103NE can be provided without a glucose-specific enzyme thatrequires more complex fabrication processes and is less stable than thesolid-state material of the nanoporous layer 117. Further, the enzymaticsensing electrodes 103NE can operate without an electron mediator thatfacilitates electron transfers between different materials. Inembodiments, the working electrode 103NE includes neither an enzyme noran electron mediator.

Materials for Nanoporous Layer

In some embodiments, the nanoporous layer 117 is made of or includesplatinum (Pt), gold (Au), palladium (Pd), rhodium (Rh), titanium (Ti),ruthenium (Ru), tin (Sn), nickel (Ni), copper (Cu), indium (In),thallium (Tl), zirconium (Zr), iridium (Ir), or an oxide of theforegoing elements, although not limited thereto. In other embodiments,the nanoporous layer 117 is made of or includes an alloy material of twoor more of the metal elements listed in the previous sentence includingPt-Ir, Pt-Ru, Pt-Pd, although not limited thereto.

Roughness Factor Defined

Roughness factor or rugosity is a ratio of a real surface area to ageometric surface area of an object. Here, the geometric surface arearefers to a projected area of the object that is projected onto a flatsurface without considering internal surfaces within the object. Thereal surface area refers to the total area of surfaces consideringinternal surfaces. Referring to FIG. 4, for example, if the nanoporouslayer 117 is in a rectangular block having a height or depth 118 and atop rectangle 119, the projected area or geometric surface area of thenanoporous layer is the area of the top rectangle that is exposed tooutside. The real surface area of a nanoporous layer may beelectrochemically measured, for example, using a well-known cyclicvoltammetric technique that detects electric current from protonadsorption on the real surface.

Roughness Factor of Nanoporous Layer

The roughness factor value indicates the total amount of internal poreswithin the nanoporous layer 117. The roughness factor of the nanoporouslayer 117 may relate to the sensitivity of the nanoporous layer 117 forthe glucose oxidation. Generally the higher the roughness factor, themore glucose oxidation may occur. The roughness factor of the nanoporouslayer 117 is at or about 100, 200, 300, 400, 500, 600, 700, 800, 900,100, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100,2200, 2300, 2400 or 2500. In embodiments, the roughness factor may bewithin a range formed by selecting any two numbers (two roughness factorvalues) listed in the immediately previous sentence, e.g., between about100 and about 2500, between about 750 and about 1250, or between about850 and about 1150.

Thickness of Nanoporous Layer

The roughness factor value does not indicate the level of porosity ordensity of the nanoporous material in its unit volume while the valuemay indicate the total amount of internal pores. Thus, depending uponthe level of porosity of the nanoporous material, in embodiments,thickness of the nanoporous layer may be adjusted to achieve a targetvalue for the roughness factor. In embodiments, the thickness ofnanoporous layer 117 may be about 0.03, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5,6, 7, 8, 9 and 10 μm. In some embodiments, the thickness may be within arange formed by selecting any two numbers (two thickness values) listedin the immediately previous sentence, e.g., between about 0.05 μm (50nm) and about 10 μm, between about 0.5 μm and about 8 μm, or betweenabout 2 μm and about 7 μm.

Morphologies

The nanoporous layer 117 may have different internal morphologies ineach specific manufacture. In some embodiments, the nanoporous layer 117may include or be made of nanoparticles deposited together formingnanopores among themselves (interparticular nanopores). In otherembodiments, the nanoporous layer 117 may include or be made of clustersof nanoparticles deposited together that form interparticular nanoporeswithin a cluster and also spaces among clusters (intercluster gaps orspaces). In other embodiments, the nanoporous layer 117 may include orbe made of repetition of a specific shape of nanostructure such ashexagonal structure that includes nanopores therein. Also, in eachspecific manufacture, the nanoporous layer 117 may have different levelsof porosity and different roughness factor values per unit volume.

Making Nanoporous Layer

The nanoporous layer 117 may be prepared using a liquid composition thatcontains metal ions and a surfactant. In embodiments, differentmorphologies of the nanoporous layer may be formed using differentphases of the surfactant. A micelle phase, a reverse micelle phase, aliquid crystalline phase or another phase of the surfactant may be usedto produce the nanoporous layer in a particular morphology. In thesedifferent phases, the metal ions are aligned or locally concentratednext to hydrophilic moieties of the surfactant. The localized metal ionsin the liquid composition are subject to additional processes forreduction and deposition on a surface to provide a nanoporous layer 117having different morphologies.

Clustered Nanoporous Layer Clustered Morphology

FIG. 5A is an illustration of a vertical cross-section of nanoporouslayer having a clustered morphology 120 over a substrate 129. Innano-sized reality, the top surface of the substrate 129 may not be asstraight as illustrated and may be bumpy. In the clustered morphology120, a number of nanoparticles 121 get together and form irregularlyshaped clusters 125. For the sake of illustration, different shadings orhatchings are used in different clusters 125. These irregularly shapedclusters 125 are stacked irregularly to form the nanoporous layer. FIG.5B is a transmission electron microscope (TEM) photographic image ofsome clusters 125 before they deposit to form a nanoporous layer. FIG.5C is a zoomed-in image of the circled portion of FIG. 5B. FIG. 5D is ascanning electron microscope (SEM) photographic image of a nanoporouslayer having a clustered morphology taken from the top of the nanoporouslayer.

Pores and Spaces of Clustered Morphology

With irregular stacking of irregularly shaped clusters 125, neighboringclusters form intercluster gaps or spaces 127 between them. Theseintercluster gaps 127 may be nano-sized and micro-sized. In thisdisclosure, nano-size means greater than 1 nm and smaller than 100 nm,and micro-size means greater than 100 nm and smaller than 100 μm. Eachcluster 125 includes or is made of generally spherical or ovalnanoparticles 121. In each cluster, individual nanoparticles aregenerally separate from one another and form small gaps 123therebetween. The small gaps are nano-sized and referred to asinterparticular nanopores 123. In embodiments, interparticular nanoporesare found throughout the clusters. In embodiments, interparticularnanopores form interconnected or networked channels within each cluster.FIGS. 5A and 5D show these interparticular nanopores 123 in each cluster125.

Forming Intercluster Gaps/Spaces

In embodiments, to produce a clustered morphology, irregularly shapedclusters 125 are first prepared as suspension in liquid. Then, thesuspension is dispensed on the substrate 129, which is subject todrying. As the liquid dries off, clusters are spontaneously depositedover the substrate and over other clusters. No external force may beapplied to the clusters while drying. Accordingly, the clusters do notget packed as they deposit. As clusters deposit and stack over otherclusters, each cluster may contact the substrate surface or neighboringclusters. After completion of drying, the clusters abut or contactadjacent or neighboring clusters. The deposited clusters areinterconnected or integrated via the abutments and contacts. Due to theirregular shapes of individual clusters, irregularly shaped gaps andspaces are formed between adjacent clusters, in which the gaps andspaces define the irregular shapes of the deposited clusters as if thesurfaces and contours of deposited clusters are surrounded by theirregularly shaped gaps and spaces. The irregularly shaped gaps andspaces are referred to as intercluster gaps or spaces 127.

Distribution of Clusters and Intercluster Gaps

In embodiments, the irregularly shaped cluster bodies 125 aredistributed throughout the clustered morphology 120 of the nanoporouslayer 117. The irregularly shaped cluster bodies 125 are interconnectedvia abutments, which means these cluster bodies contact themselves andform a three-dimensional network of cluster bodies generally throughoutthe nanoporous layer 117. The intercluster gaps 127 define and surroundsurfaces of the irregularly shaped cluster bodies and are interconnectedthemselves to form a three-dimensional interconnected or networkedchannels throughout the nanoporous layer 117. The intercluster gaps andspaces 127 are well distributed throughout the nanoporous layer 117 fromthe top (not shown) to the bottom (on or immediately above the substrate129). The three-dimensional network of irregularly shaped clusteredbodies and the three-dimensional network of irregularly shaped gapsthree-dimensionally are complementary to form a highly networkedthree-dimensional mesh structure. The three-dimensional network ofcluster bodies and channels may be similar to the three-dimensionalinternal shapes of a sponge except that the interparticular gaps andspaces are networked together throughout the nanoporous layer 117.

Distribution of Nanoparticles and Interparticular Nanopores

Given that each cluster is formed with many nanoparticles 121 andinterparticular nanopores 123, the nanoparticles 121 and interparticularnanopores 123 are distributed generally throughout the nanoporous layer117. Accordingly, interparticular nanopores 123 are interconnectedwithin each cluster and interconnected with interparticular nanopores ofother clusters generally throughout the nanoporous layer 117 viainterparticular nanopores in abutments between clusters and viaintercluster gaps 127 that are interconnected throughout the nanoporouslayer 117.

Intercluster Gaps/Spaces for Diffusion of Glucose

In embodiments, the interconnection of intercluster gaps 127 providesnetworked channels for diffusion of glucose molecules (0.7-0.8 nm long)within the nanoporous layer 117. It is understood that glucose oxidationoccurs primarily in nano-sized interparticular nanopores rather than inmicro-sized spaces. As the intercluster gaps 127 are networked orinterconnected throughout the nanoporous layer 117, glucose moleculesmay reach almost anywhere in the nanoporous layer 117 via theintercluster spaces that are large scale considering the size of glucosemolecules. Also, as the intercluster gaps 127 are well interconnected tothe interparticular nanopores 123, interparticular nanopores 123anywhere in the nanoporous layer 117 may be exposed and open for glucoseoxidation. Accordingly, the three-dimensional interconnected ornetworked channels of the intercluster gaps may provide more glucoseoxidation, i.e., stronger signals (higher electric current) of theglucose oxidation than a nanoporous layer without such interconnectedchannels formed of intercluster gaps.

Two Types of Particles and Two Types of Pores

As discussed, the clustered morphology 120 includes two different typesof particles defining two different types of pores. In terms ofparticles, one is the nanoparticles 121, and the other is the clusters125 made of nanoparticles 121. In terms of pores, one is theinterparticular nanopores 123 between nanoparticles 121 within a cluster125, and the other is the intercluster gaps 127 between clusters 125.

Clusters of Nanoparticles

The TEM photographic image of FIG. 5B shows clusters in irregularshapes. The number of nanoparticles 121 in each cluster may vary wildly,and the size of clusters 125 may vary accordingly. In clusteredmorphologies, some clusters 125 are nano-sized (smaller than 100 nm),and others are micro-sized (100 nm to 100 μm). The clusters 125 have alength or diameter of about 20, 40, 60, 80, 100, 120, 140, 160, 180,200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460,480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680 or 700 nm. Inembodiments, the length or diameter of the clusters 125 may be within arange formed by selecting any two numbers (two length or diametervalues) listed in the immediately previous sentence, e.g., between about20 nm and about 300 nm, or between about 60 nm and about 240 nm. Theclusters 125 may have a mean diameter or length of about 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,230, 240, 250, 260, 280 or 300 nm. In embodiments, the mean diameter ofthe clusters 125 may be within a range formed by selecting any twonumbers listed in the immediately previous sentence, e.g., between about100 nm and about 220 nm.

Nanoparticles

The TEM photographic image of FIG. 5C shows nanoparticles in a singlecluster. The nanoparticles 121 in the cluster are discrete and generallyin a spherical (ball-like) or oval (egg-like) shape, although notlimited thereto. The nanoparticles 121 have a diameter of about 1.0,1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or 6.5. Inembodiments, the diameter may be within a range formed by selecting anytwo numbers (two diameter values) listed in the immediately previoussentence, e.g., between about 2 nm and about 5 nm. The nanoparticles 121may have a mean diameter of about 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5,3.75 or 4.0. In embodiments, the mean diameter of the nanoparticles 121may be within a range formed by selecting any two numbers listed in theimmediately previous sentence, e.g., between about 2.5 nm and about 4.0nm, between about 2.75 nm and about 3.75 nm, or between about 2.25 nmand about 3.5 nm. In embodiments, nanoparticles having a mean diameterof 2-5 nm are found throughout the nanoporous layer 117.

Interparticular Nanopores

The TEM photographic image of FIG. 5C also shows interparticularnanopores between nanoparticles in the cluster. The interparticularnanopores are networked and interconnected within the cluster. Theinterparticular gaps or nanopores 123 have an interparticular gapdistance between two immediately neighboring nanoparticles within thesame cluster. The interparticular gap distance is about 0.25, 0.5, 0.75,1.0, 1.25, 1.5, 1.75, 2.0, 2.5, 3.0, 3.5, 4.0 or 4.5 nm. In embodiments,the interparticular gap distance may be within a range formed byselecting any two numbers (two distance values) listed in theimmediately previous sentence, e.g., between about 0.5 nm and about 4.5nm, or between about 1.5 nm and about 4.0 nm. The interparticularnanopores 123 may have a mean interparticular gap distance of about 0.5,0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0 or 3.5 nm. Inembodiments, the mean interparticular gap distance of the nanopores 123may be within a range formed by selecting any two numbers listed in theimmediately previous sentence, e.g., between about 0.75 nm and about 1.5nm, or between about 1.0 nm and about 2.5 nm. In embodiments,interparticular nanopores 123 having a mean interparticular gap distanceof 1-2.5 nm are found throughout the nanoporous layer 117.

Intercluster Gaps/Spaces

The SEM photographic image of FIG. 5D shows openings of the networkedintercluster gaps that can be seen from the top of the nanoporous layer.Although the three-dimensional shapes are not well presented in thetwo-dimensional image of FIG. 5D, the top surface of nanoporous layerincludes valleys and hills formed by stacked clusters. Inside thenanoporous layer, the valleys and hills form the intercluster gaps. Theintercluster gaps or spaces are in irregular shapes. The interclustergaps 127 are nano-sized to micro-sized. The intercluster gaps 127 havean intercluster gap distance of about 25, 50, 75, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525,550, 575, 600, 625, 650, 675 or 700 nm. In embodiments, the interclustergap distance may be within a range formed by selecting any two numberslisted in the immediately previous sentence, e.g., between about 100 nmand about 1000 nm. The intercluster gaps 127 have a mean interclustergap distance of about 100, 150, 200, 250, 300, 350, 400, 450 or 500 nm.In embodiments, the mean intercluster gap distance may be within a rangeformed by selecting any two numbers listed in the immediately previoussentence, e.g., between about 150 nm and about 400 nm.

Making Clustered Nanoporous Layer Overall Process

In embodiments, a nanoporous layer having a clustered morphology may beprepared using an isotropic reverse micelle phase (or “reverse micellephase”)” of a surfactant. Referring to FIG. 6A, at step 601, an aqueousliquid composition is prepared with a metal ion source and a surfactantin a reverse micelle phase. The metal ions are locally concentratedwithin hydrophilic spaces of individual reverse micelles. Subsequentlyat step 603, a reducing agent is added to the reverse micelle phase toform metal nanoparticles dispersed in the liquid composition containingthe surfactant (“nanoparticle colloid” or “nanoparticle-surfactantcolloid”). Subsequently at step 605, the surfactant is removed from thenanoparticle-surfactant colloid, and the nanoparticle clusters dispersedin liquid (“cluster colloid” or “cluster-liquid colloid”) are collected.Optionally at step 607, the collected cluster colloid is mixed with anon-surfactant liquid. At step 609, the cluster colloid is dispensedonto a surface, for example, by printing technique without use ofelectroplating. Subsequently at step 611, the liquid is dried off toform a nanoporous layer 117 on the surface 129.

Surfactant

Surfactants are amphiphilic organic compounds having a hydrophilic head(or hydrophilic moiety) and a hydrophobic tail (hydrophobic moiety) in asingle molecule. Surfactants may form different structures or phases inwater depending on the concentration and temperature. FIG. 7 is anexample phase diagram of a surfactant showing different phases includingmicelle phase 131, hexagonal phase 133, Lamellae phase 135, and twomicelles phases 137.

Preparing Isotropic Reverse Micelle Phase

At step 601, an isotropic reverse micelle phase is prepared with anaqueous liquid composition containing a surfactant, metal ions andwater. As in the conceptual illustration of FIG. 8, the reverse micellephase includes reverse micelles 141 formed by the surfactant molecules.Each reverse micelle 141 includes a hydrophilic core 143 surrounded byhydrophobic tails radiating from the hydrophilic core. The hydrophiliccore 143 encloses hydrophilic components of the liquid composition,i.e., water and metal ions. Thus, the metal ions are locallyconcentrated within the hydrophilic core 143 of reverse micelles.

Surfactant Examples

The surfactant is chosen from those that can form an isotropic reversemicelle phase under reasonable conditions for processing. In someembodiments, a non-ionic surfactant is used, although not limitedthereto. Non-limiting examples of the surfactant includealkylbenzenesulphonates, alkyl-polyglycoside, alkyl sulphates,carboxylates, carboxylic esters, Cetomacrogol 1000™, cetostearylalcohol, cetyl alcohol, cocamide DEA, cocamide MEA, decyl glucoside,decyl polyglucose, disodium cocoamphodiacetate, ethoxylated aliphaticalcohol, glycerol monostearate, glycol esters of fatty acids, IGEPALCA-630™, isoceteth-20, lauryl glucoside, maltosides, monolaurin,mycosubtilin, naphthalenesulphonates, narrow-range ethoxylate, NonidetP-40™, nonoxynol-9, nonoxynols, NP-40™, octaethylene glycol monododecylether, N-Octyl beta-D-thioglucopyranoside, octyl glucoside, oleylalcohol, PEG-10 sunflower glycerides, pentaethylene glycol monododecylether, polidocanol, poloxamer, poloxamer 407, polyethoxylated tallowamine, polyethylene glycol esters, polyglycerol polyricinoleate,polyoxyethylene fatty acid amides, polyoxyethylene surfactants,polysorbate, polysorbate 20, polysorbate 80, sorbitan, sorbitanmonolaurate, sorbitan monostearate, sorbitan tristearate, stearylalcohol, surfactin, sulphated alkanolamides, sulphonates, Triton X-100™,and Tween 80™. A skilled artisan in the relevant field would appreciatewhat would constitute the reasonable conditions.

Conditions for Reverse Micelle Phase

Subsequent to choosing the surfactant, its concentration and thetemperature are adjusted to form an isotropic reverse micelle phase. Thesurfactant' s concentration and temperature may be determined withreference to the surfactant's phase diagram. When the phase diagram isnot available, some experiments for finding appropriate concentrationand temperature may be necessary using known laboratory techniques andprocedures. For example, when Triton X-100™ is used for the surfactant,the concentration of 10-60 wt % and temperature of 40-80° C. may providethe reverse micelle phase.

Source of Metal Ions

One or more metal ions corresponding to the metal or alloy for thenanoporous layer are chosen for the liquid composition. The metal ionsare added in the form of a compound containing the ionic metal such asan acid, base or salt. Non-limiting examples of the metal sourcecompound include H₂PtCl₆, H₂Pt(OH)₆, H₂PtCl₂(OH)₄, H₂Pt(SO₄)(OH)₄,PtCl₄, K₂PtCl₆, PdCl₂, and TiCl₄.

Concentration of Metal Ions

The concentration of metal ions is also adjusted for best performance.When the concentration is too low, nanoparticles may not be formed. Whenthe concentration is too high, it may affect the formation or stabilityof the reverse micelle phase of the surfactant. The concentration ofmetal ions is about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007,0.008, 0.009, 0.01, 0.012, 0.014, 0.016, 0.018, 0.02, 0.022, 0.024,0.026, 0.028, 0.03, 0.032, 0.034, 0.036, 0.038, 0.04, 0.042, 0.044,0.046, 0.048, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09,0.095 or 0.1 M. In embodiments, the concentration may be within a rangeformed by selecting any two numbers (two molarity values) listed in theimmediately previous sentence, e.g., between about 0.01 and about 0.03M, between about 0.02 and 0.03 M, etc. Within an appropriateconcentration range, it has been observed that the level ofconcentration affects the speed of formation of nanoparticles.

Different from Plating Bath

The reverse micelle phase prepared at step 601 is not a plating bathcomposition for electroplating. Unlike in the plating bath, no metalchelating agent may be needed.

Forming Nanoparticles

At step 603, a reducing agent is mixed to the aqueous liquid compositionin the reverse micelle phase. When the reducing agent enters thehydrophilic core 143 of reverse micelles 141, it reduces metal ions tometal atoms inside the hydrophilic core 143. Because the metal ions arelocally concentrated inside the hydrophilic cores 143, initially metalatoms remain inside the hydrophilic cores 143. The metal atoms insideeach hydrophilic core 143 coagulate together and grow to form metalnanoparticles. One metal nanoparticle may grow from one reverse micelle,although not limited thereto. The resulting metal nanoparticles aregenerally not charged, i.e., neutral. However, some nanoparticles may beslightly positively charged on their surfaces. Thus far, no electricityis applied to form the metal nanoparticles.

Nanoparticle Colloid

The nanoparticles are dispersed in the liquid to provide a nanoparticlecolloid. FIG. 8 conceptually illustrates the resulting nanoparticlecolloid. In the course of the reduction of metal ions and growth ofnanoparticles, some reverse micelles break or burst, and accordinglynanoparticles from those burst reverse micelles may be dispersed intothe hydrophobic space. Some of those nanoparticles 151 may freely floatin the resulting colloid composition outside hydrophilic cores ofreverse micelles. Some other nanoparticles 153 may be surrounded orbound by hydrophilic heads of surfactant molecules outside hydrophiliccores of reverse micelles. Some nanoparticles 155 remain inside reversemicelles 141. Overall, in the resulting nanoparticle colloid, the solidnanoparticles 151, 153, 155 are dispersed in the liquid compositionincluding reverse micelles 141, water and surfactant molecules. Becausethe nanoparticles 151, 153, 155 are significantly separated from eachother in the nanoparticle colloid composition, it is unlikely that thenanoparticles congregate and grow to larger particles.

Reducing Agent

The reducing agent is a chemical entity that can donate one or moreelectrons to the metal ions contained in the nanoparticle colloid. Thereducing agent is a hydrophilic compound for entering the hydrophiliccore of the reverse micelle. Non-limiting examples of the hydrophilicreducing agent include ascorbic acid, acetic acid, form aldehyde, citricacid, hydroxylamine, hypophosphite, etc.

Amount of Reducing Agent

The hydrophilic reducing agent is added to the nanoparticle colloid inan amount sufficient to reduce the metal ions contained therein. In someembodiments, the reducing agent is added in an excessive amount that issubstantially more than the stoichiometric amount for reducing the totalmetal ions contained in the nanoparticle colloid. Here “substantiallymore than” means more than by 20, 40, 60, 80, 100, 120, 140, 160, 180,200, 250, 300 or 400%.

Stirring

While and/or after adding the reducing agent, the mixture may be stirredto facilitate distribution of the reducing agent. Stirring mayfacilitate the reducing agent to enter the hydrophilic spaces of thereverse micelles. Accordingly, the time for fully reducing the metalions in the hydrophilic spaces can be reduced. Stirring may be performedcontinuously or intermittently. In embodiments, stirring is performedfor a period between 1 hour and 10 hours.

Removing the Surfactant and Forming Clusters

At step 605, the surfactant is substantially removed from thenanoparticle colloid composition to form clusters of nanoparticles. Inthe nanoparticle colloid, the surfactant may stabilize individualnanoparticles, and accordingly nanoparticles may not cluster togetherwhen a significant amount the surfactant is present. To remove thesurfactant from the nanoparticles, the nanoparticle colloid is subjectto centrifugation. After the centrifugation, most nanoparticles settlein the bottom portion, and the surfactant molecules may be in thesupernatant and in the bottom portion. The supernatant is separated fromthe bottom portion containing most of the nanoparticles. In embodiments,liquid may be added to the separated nanoparticles to dilute thesurfactant in the collected bottom portion. The liquid added to thenanoparticles may be water or aqueous solution, which may be an acidicor basic solution although not limited thereto. The centrifugation,collecting the bottom portion, and adding liquid may be repeatedmultiple times to collect nanoparticles in which the surfactant issubstantially removed.

Chemical Bond between Surfactant and Nanoparticle

Depending upon the surfactant, some nanoparticles have a strong chemicalbond with hydrophilic heads of some surfactant molecules. Surfactantmolecules having negatively charged hydrophilic heads may form acoordinate bond with nanoparticle surfaces. Also, if the surfactantmolecules have electron-abundant hydrophilic heads (even if they are notcharged), they may form a coordinate bonding with nanoparticle surfaces.When such surfactants are used, the chemical bond must be broken toremove the surfactant from the nanoparticle colloid.

Breaking Chemical Bond

In some embodiments, an acidic or basic solution is added to thenanoparticles-surfactant colloid after forming nanoparticles at step 603and before centrifugation at step 604 of FIG. 6B. The acid or base ofthe added solution causes a chemical reaction to break the coordinatebond between the surfactant and nanoparticles to free nanoparticles. Forexample, protons from acid may bond with the negatively charged orelectron-abundant surfactant heads to free the nanoparticles. Thesubsequent centrifugation and collection of bottom portion separate thenanoparticles freed from the surfactant molecules. In embodiments,adding acidic or basic solution may be performed at least once beforecentrifugation. In some embodiments, adding acidic or basic solution maybe performed before each centrifugation. In embodiments, the acid andbase may be washed with water or other solvent after centrifugation.

Acidic or Basic Solution

In embodiments, the acid or base is chosen in view of the surfactantsuch that the surfactant molecules are effectively detached from thenanoparticles. In embodiments, the acidic solution has a pH value lowerthan about 3, although not limited thereto. For example, non-limitingexamples of acid for the acidic solution include HCl, HNO₃, H₂SO₄,HClO₄, etc. In embodiments, the basic solution has a pH value higherthan about 10, although not limited thereto. For example, non-limitingexamples of base for the basic solution include NaOH, KOH, Ca(OH)₂, etc.

Cluster Colloid

After or in the processes for removing the surfactant and collectingnanoparticles, nanoparticles tend to cluster together or agglomerate toform clusters of nanoparticles. In liquid, the clusters are dispersed toform a cluster colloid. Each cluster includes and is made of the metalnanoparticles interacting with each other to form a larger body.Individual nanoparticles in the clusters are most likely electricallyneutral. Although the invention is not bound by any theory or belief, itis believed that protons, hydroxide and other charged electrolytes maybe bound to nanoparticles surfaces and that ionic interactions of theseelectrolytes with adjacent nanoparticles may keep neighboringnanoparticles together to form the clusters. In fact, the liquid of thecluster colloid contains a good amount of electrolytes originated fromthe metal ion source and the acidic or basic solution used in theprevious preparation steps although the surfactant molecules weresubstantially removed.

Clusters and Nanoparticles

FIG. 9 provides TEM photographic images of nanoparticle clusters from adiluted sample of cluster colloid. Two of the images of FIG. 9 are alsofound in FIGS. 5B and 5C. In these images, the clusters do not have aregular shape and are about 30 to about 500 nm long. The nanoparticles121 in the clusters are discrete and generally spherical or oval, andhave a diameter of about 2-3 nm. There are interparticular gaps 125between neighboring or adjacent nanoparticles 121 with a gap distance ofabout 1-2 nm. These interparticular nanopores 125 are primarilyresponsible for glucose oxidation in a glucose-sensing electrode havinga clustered nanoporous layer.

Centrifugation

The centrifugation may be performed at a rotational speed between 3000and 5000 rpm. The centrifugation may continue for a period between 3 and15 minutes. After centrifugation, the supernatant is removed, and thebottom portion containing the nanoparticles are collected. Liquid isadded to the collected bottom portion to dilute surfactant containedtherein. The centrifugation, collecting bottom portion and adding liquidmay be repeated multiple times, e.g., three times or more.

Surfactant Substantially Removed

With the multiple processing of centrifugation, the surfactant issubstantially removed. In the resulting cluster colloid, theconcentration of surfactant becomes significantly low although it maynot be completely removed. In the beginning, the reverse micelle phasecontains the surfactant from about 10 to about 60 wt. The resultingcluster colloid may contain no surfactant at all. Practically, theresulting cluster colloid is substantially free of the surfactant. Theremaining surfactant in the resulting cluster colloid or in the finalcollection of bottom portion may be greater than 0.0001 parts by weightwith reference to 100 parts by weight for the nanoparticles and smallerthan about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.6 parts by weight withreference to 100 parts by weight for the nanoparticles. In embodiments,the remaining surfactant may be in an amount smaller than about 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4 or0.5 parts by weight with reference to 100 parts by weight for thenanoparticles.

Concentration of Nanoparticles in Cluster Colloid

After the multiple processing of centrifugation, the total amount ofnanoparticles (as part of clusters and free nanoparticles) in the finalcollection of bottom portion may be about 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39 or 40 wt %. In embodiments, the concentration may bewithin a range formed by selecting any two numbers listed in theimmediately previous sentence, e.g., between about 20 and about 30 wt %,between about 15 and 25%, etc.

Storing Cluster Colloid

The clusters are dispersed in the cluster colloid for an extendedperiod, e.g., longer than a week or a month without any treatment. Thecluster colloid may be stored in a container for a while afterpreparation and before subsequent processing. Once prepared, the clustercolloid may be subject to sales and transportation for processing byothers or in other locations. To maintain the colloidal property for alonger period, the concentration of nanoparticles may be adjusted afterthe final collection of bottom portion. In embodiments, the clustercolloid of the final collection of bottom portion may be stored ortransported in a container with or without adjusting the concentration.

Adjusting Concentration for Dispensing

At step 607, the collected cluster colloid may be stored for a whilewith or without dilution with a solvent. The dilution may be to adjustthe concentration of clusters in the cluster colloid for the subsequentprocessing, e.g., dispensing. The solvent may be water or organiccompound. One or more additive compounds may be added. By the dilution,the concentration of the nanoparticles or clusters is adjusted to about0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3,3.1, 3,2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14 or 15 wt %. In embodiments, theconcentration of the nanoparticles or clusters may be within a rangeformed by selecting any two numbers listed in the immediately previoussentence, e.g., between about 0.5 and about 2 wt %, between about 1 and3 wt %, etc. After the dilution, the remaining surfactant may be lessthan about 0.1, 0.2, 0.4, 0.6, 0.8 1, 1.2, 1.4, 1.6, 1.8 or 2 wt %.

Dispensing Cluster Colloid

At step 609, the cluster colloid is dispensed on a substrate 129 forproducing the nanoporous layer, while it maintains its colloidalproperty. Various dispensing technologies may be utilized to dispensethe cluster colloid. Dispensing may be controlled to form a certainthickness of the dispensed cluster colloid or to provide an appropriatethickness of the resulting nanoporous layer after subsequent drying. Inthe alternative, dispensing may be controlled to provide an appropriateroughness factor value of the resulting nanoporous layer.

Underlying Substrate

The cluster colloid may be applied onto a substrate made of anymaterial. In embodiments for glucose-sensing electrodes, the clustercolloid may be applied onto a conductive or semiconductive surface forthe conductive layer 110 as discussed above. In some embodiments, thesubstrate includes two or more conductive layers.

Drying Liquid to Form Clustered Nanoporous Layer

At step 611, the dispensed cluster colloid is subject to a condition fordrying the liquid. Upon dispensing, the nanoparticle clusters are floatin the liquid and freely travel horizontally and vertically. As theliquid dries off, the height of the cluster colloid decreases. As theliquid continues to dry off, clusters become contacting neighboringclusters vertically between the underlying substrate 129 and the top ofthe cluster colloid and horizontally. Mobility of the clusters becomessignificantly limited. Sometime later, the liquid level becomes lowerthan clusters located at or near the top. Once the drying is complete,the nanoparticle clusters deposited on the substrate 129 forming ananoporous layer having a clustered morphology 120 as illustrated inFIG. 5A.

Thickness of Nanoporous Layer

The resulting nanoporous layer has a thickness of about 0.4, 0.6, 0.8,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 μm. In embodiments, thethickness may be within a range formed by selecting any two numberslisted in the immediately previous sentence, e.g., between about 1 μmand about 10 nm.

No Washing Nanoporous Layer

The resulting nanoporous layer does not require washing with water orother liquid. In embodiments, the resulting nanoporous layer in aclustered morphology is not washed with water or other liquid at allsubsequent to drying. In embodiments, the nanoporous layer is notsubject to contacting liquid except in a subsequent processing foradding a layer over the nanoporous layer.

Yield—Recovery of Metal

If an excessive amount of reducing agent is added to the nanoparticlecolloid, most metal ions therein are reduced to form metal atoms, whichcoagulate to form nanoparticles. The subsequent processing of removingthe surfactant also collects most nanoparticles in clusters. Thus, mostmetal ions added to the foregoing processes are ultimately collected inthe form of clusters of nanoparticles and deposited in the resultingnanoporous layer 117. In embodiments, over 89, 90, 91, 92, 93, 94, 95,96, 97 or 98% of inputted metal ions are collected in the form ofnanoparticle clusters before dispensing.

Mass Production

The nanoporous layer 117 can be mass-produced by printing the clustercolloid over the substrate 129. Printing the cluster colloid takes onlyone second or two. While drying the liquid may take longer time, it onlytakes a large space for drying. In embodiments, a number of separatesubstrates are provided, and printing may be performed on each of theseparate substrates. Then, each printed substrate is dried to form ananoporous layer. Alternatively, multiple areas are printed with thecluster colloid on a single substrate, and the single substrate may besubsequently cut into multiple pieces, each including a printed area.The single substrate may be dried before cutting.

No Electroplating or No Application of Electricity

Throughout the process, no electroplating is utilized to form theclustered morphology for the nanoporous layer. Further, no electricityis applied to the substrate 129 on which the nanoporous layer is formed.

Non-Clustered Nanoporous Layer Non-Clustered Morphology

FIG. 10A illustrates a non-clustered morphology 161 for the nanoporouslayer 117. As in the clustered morphology 120, the non-clusteredmorphology 161 includes both nanoparticles 121 and interparticularnanopores 123 formed between neighboring or adjacent nanoparticles 121.The discussions of the nanoparticles 121 and interparticular nanopores123 generally apply to the non-clustered morphology 161. FIG. 10B is aTEM photographic image of a non-clustered morphology of a nanoporouslayer formed on a metal surface, in which the dark portion is part ofthe metal surface. The nanoparticles of and interparticular pores in theTEM photographic image are similar to those in the illustration of FIG.10A.

No Clusters and No Intercluster Gaps

Unlike the clustered morphology 120, the non-clustered morphology 161does not include clusters 123 or intercluster gaps 127. To produce anon-clustered morphology, nanoparticles are deposited on the substrate129 by electroplating without preparing clusters before electroplating.As a result, neither clusters nor intercluster gaps are formed in theresulting configuration, i.e., non-clustered morphology 161.Accordingly, the non-clustered morphology 161 does not have thecharacteristics of the clustered morphology coming from the clusters 123or intercluster gaps 127.

Cavities of Non-Clustered Morphology

While no intercluster gaps exist, the non-clustered morphology 161 mayinclude internal cavities 133 that are significantly larger than theinterparticular nanopores 123. The internal cavities 133 may be formedduring the course of electroplating because nanoparticles are not alwayssequentially stacked on the immediately underlying surface. The internalcavities 133 are in irregular shapes and in irregular sizes. Theinternal cavities 133 may be found throughout the nanoporous layer 117.

Cavities Distinguished from Intercluster Gaps or Spaces

The cavities 133 of the non-clustered morphology are distinguished fromthe intercluster gaps 127 of the clustered morphology 120. The cavities133 are formed because electroplating and deposition of nanoparticlesare not at the same rate over surfaces of the substrate 129. Thecavities 133 do not surround or define a cluster or clusters 125 ofnanoparticles 121. Rather, each cavity 133 is surrounded or defined bythe agglomerated or conglomerated body of nanoparticles 121. While thecavities 133 may be interconnected via interparticular nanopores 123,the cavities 133 themselves are not interconnected throughout thenanoporous layer 117 or some substantial portion thereof. Further, thecavities 133 do not occupy as much volume of the nanoporous layer 117(lower roughness factor in the non-clustered morphology) as theintercluster gaps 127 (higher roughness factor in the clusteredmorphology).

Substrate Substantially Covered with Nanoparticles

Referring to FIGS. 10A and 10B, the top surface of the substrate 129 issubstantially covered with nanoparticles 121. In some embodiments, nosubstantial internal spaces are formed on or immediately above thesubstrate 129, although not limited thereto.

Clustered and Non-Clustered Morphologies Compared

Overall, the clustered morphology 120 is much less dense than thenon-clustered morphology 161. For the same thickness, the clusteredmorphology 120 has a higher roughness factor than the non-clusteredmorphology 161, and accordingly, to produce the same roughness factor,the clustered morphology 120 may be thinner than the non-clusteredmorphology. Also, given that the cluster's irregular shapes,intercluster gaps 127 of the clustered morphology 120 are interconnectedgenerally throughout the nanoporous layer 117, whereas the internalcavities 133 of the non-clustered morphology 161 are not as connected asthe intercluster gaps 127. Accordingly, interparticular nanopores 125within clusters 123 are connected to the network of intercluster gaps127 in the clustered morphology 120, whereas in the absence ofintercluster gaps in the non-clustered morphology 161, interparticularnanopores 125 may not be as connected as those in the clusteredmorphology 120.

Making Non-Clustered Nanoporous Layer—Electroplating Overall Process

A nanoporous layer having a non-clustered morphology may be preparedusing an electroplating. Referring to FIG. 11, at step 1101, a platingbath is prepared to contain metal ions and a surfactant in a reversemicelle phase. Subsequently at step 1103, electroplating is performed inthe plating bath for depositing a nanoporous layer in the non-clusteredmorphology. At step 1105, the resulting nanoporous layer is washed toremove surfactant therefrom.

Preparing Plating Bath

At step 1101, a plating bath is similar to the reverse micelle phase ofstep 601 of FIG. 6A for making a clustered nanoporous layer withoutelectroplating. The plating bath includes a surfactant in reversemicelle phase and a metal ion source material as in making a clusterednanoporous layer. All discussions relating to the surfactant and metalion source material of step 601 of FIG. 6A are applicable to the step1101 of FIG. 11. However, the plating bath at step 1101 is not identicalto the reverse micelle phase of step 601. One important difference maybe that the plating bath may require some additional materials in viewof the electroplating in the next step. For many metal source compoundsthat may spontaneously be reduced, the plating bath may require achelating agent to keep the metal ions from being spontaneously reducedduring and before electroplating. In contrast, no such chelating agentmay be needed in the reverse micelle phase of step 601.

Electroplating

At step 1103, electroplating is performed in the aqueous liquidcomposition of reverse micelle phase containing metal ions. In a platingbath containing the liquid composition, cathode and anode electrodes aresubmerged and are connected to a power supply. When a DC voltage isapplied between the cathode and anode electrodes, the cathode electrodesupplies electrons to the aqueous liquid composition. Electrons may jumpfrom the cathode electrode to nearby hydrophilic spaces of reversemicelles to reduce the positively charged metal ions to metal atomsinside the hydrophilic spaces. The metal atoms get together and form ametal particle, which may deposit onto the cathode electrode surface. Inthe course the reverse micelles may burst. Electrons supplied to thecathode electrode travel through the deposited nanoparticles and becomeavailable on outer surfaces of deposited nanoparticles. The electronsthen are available for reducing nearby metal ions to form metalnanoparticles for depositing over the already deposited nanoparticles.

Time for Electroplating

The electroplating is performed for about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55 or 60 minutes to obtain a nanoporous layer having roughnessfactor of 100 to 800. In embodiments, the time for electroplating may bewithin a range formed by selecting any two numbers listed in theimmediately previous sentence, e.g., between about 10 and about 30minutes. In embodiments, time for electroplating is controlled forobtaining a nanoporous layer having roughness factor of 100 or above.

Forming Layer after Layer and Cavities

In the reduction by electroplating, nanoparticles adjacent to thecathode electrode are first deposited on the surface of the cathode.Then, additional nanoparticles deposit over the previously depositednanoparticles 121. Accordingly, nanoparticles deposit generally layerafter layer over the cathode electrode. However, because depositingnanoparticles may not occur at the same rate throughout the cathodesurface and the previously deposited layer of nanoparticles, internalcavities 133 may be formed in the resulting nanoporous layer. Thedeposition of nanoparticles may grow horizontally or laterally over aspace where nanoparticles are not deposited, some cavities 133 may beenclosed with nanoparticles formed thereover. Although cavities 133 maybe ultimately interconnected via interparticular nanopores 125,micro-sized channels are not formed throughout the nanoporous layer 117or in a substantial portion thereof to interconnect the cavities 133.

Surfactant Deposited Together

In the course of electroplating, reverse micelles enclosing thesenanoparticles may burst, and the nanoparticles are deposited on thecathode electrode. A significant amount of surfactant molecules from theburst reverse micelles are deposited on the cathode electrode along withthe nanoparticles. In the course of electroplating, surfactant moleculesmay bond to nanoparticle surfaces, and nanoparticle-surfactant moleculecomplexes may be deposited together. The surfactant molecules may beinserted or trapped between nanoparticles in the resultingnanostructure.

Remaining Surfactant and Effects

The surfactant molecules deposited together with nanoparticles mayoccupy gaps and spaces between nanoparticles, i.e., interparticularpores. These surfactant molecules may effectively block nanopores andnanoparticle surfaces that are responsible for glucose oxidation.Further, the surfactant molecules may be degraded on the metal surfaces,which may contaminate the nanoparticle surfaces. Overall, thesensitivity of glucose oxidation may be affected by the surfactantremaining in the nanoporous layer.

Washing

At step 1105, the resulting nanoporous layer is washed with water orother liquid to remove surfactant molecules therefrom. However, washingis not effective to substantially remove surfactant molecules as manysurfactant molecules are trapped between neighboring nanoparticles andalso washing liquid may reach only to a certain level.

No Nanoparticle Colloid

In the electroplating method, no reducing agent is added to reduce themetal ions to form nanoparticles. During the course of electroplating,nanoparticles may be formed in hydrophilic spaces of reverse micellesthat are next to or near the cathode electrode surfaces. Thenanoparticles are then likely deposited onto the cathode electrode.However, nanoparticles are not formed in hydrophilic spaces of reversemicelles throughout the liquid composition. Accordingly, no nanoparticlecolloid is formed as illustrated in FIG. 8.

No Clusters and No Cluster Colloid

In the electroplating method, there is no step for removing thesurfactant after forming nanoparticles. Rather, the surfactant andnanoparticles are deposited together during the process ofelectroplating. Accordingly, no clusters are formed in any stage of theprocess, and no cluster colloid is formed either.

Yield—Recovery of Metal

At the completion of electroplating, the plating bath contains asignificant amount of metal ions. Thus, the recovery of metal in theelectroplating method may not be as high as in the reduction by addingan excessive amount of reducing agent as in the process for clusterednanoporous layer.

Making Nanoporous Layer Using Liquid Crystalline Phase

The nanoporous metal layer may be fabricated from a liquid crystallinephase of a surfactant. Referring to FIG. 12, at step 1201, an aqueousliquid composition is prepared to contain metal ions and a surfactant ina liquid crystalline phase, for example, in a hexagonal arrangement.Subsequently at step 1203, the aqueous liquid composition is subject toelectroplating for depositing nanoporous layer in which metal atoms aredeposited using the liquid crystalline phase as a template. At step1205, the surfactant is removed from the deposited hexagonalnanostructure. FIG. 13A illustrates formation of a hexagonalarrangement. FIG. 13B illustrates deposition of metal using a hexagonalarrangement of liquid crystalline phase.

Maltose-Blocking Layer Maltose

Maltose is a disaccharide composed of two units of glucose asillustrated in FIG. 20. Maltose may be present in blood or other bodilyfluid of human or animal. The presence of maltose in a test fluid mayinterfere with the accurate sensing of a glucose level in both enzymaticand non-enzymatic glucose-sensing systems.

Interference of Maltose in Enzymatic Glucose Sensing

Some enzymes used in enzymatic glucose-sensing system oxidize maltose aswell as glucose. Accordingly, when maltose exists in the test fluid, theenzymatic glucose-sensing system may have an inaccurate reading ofglucose level due to maltose. If an inaccurate reading is used tocontrol or adjust insulin infusion, the consequence may be serious.

Interference of Maltose in Non-enzymatic Glucose Sensing

The nanoporous layer 117 of the working electrode 103NE can oxidizemaltose at the same bias voltage for sensing glucose. With the length ofabout 1.4-1.6 nm as illustrated in FIG. 20, maltose molecules may enterinterparticular nanopores 123 of the nanoporous layer 117 and beoxidized there along with glucose. Example 9.11 and FIG. 18 confirm thatmaltose can be detected along with glucose and other interferingchemical entities in PBS. Also, Example 10.9 and FIG. 19 confirm thatmaltose can be detected along with glucose and other interferingchemical entities in serum.

Non-enzymatic Working Electrode with Maltose-Blocking Layer

Referring to FIG. 21, the working electrode 103NE includes a nanoporouslayer 117 and a maltose-blocking or maltose-screening layer 301 over thenanoporous layer 117. In embodiments, the nanoporous layer 117 iscapable of oxidizing both maltose and glucose whether it includesclustered or non-clustered morphology. The maltose-blocking layer 301may contact the underlying nanoporous layer 117 or may be separated byan intervening layer. The working electrode 103NE may also include anadditional functional layer 112 over the maltose-blocking layer 301. Inthe alternative, the additional functional layer 112 may be interposedbetween the maltose-blocking layer 301 and the nanoporous layer 117.

Selective Blocking of Maltose

The maltose-blocking layer 301 effectively or substantially blocks orinhibits maltose molecules from passing or penetrating therethroughwhile allowing glucose molecules to pass therethrough. With themaltose-blocking layer 301, maltose molecules contained in the testfluid may not reach its underlying nanoporous layer 117 at all or at asignificant concentration to interfere glucose sensing. Given theselective maltose blocking effect of the maltose-blocking layer 301, itis unlikely that the existence of maltose in the test fluid affects theglucose sensing even if the nanoporous layer 117 is capable of oxidizingmaltose at the same bias voltage for glucose oxidation. In addition, themaltose-blocking layer 301 effectively block or inhibit other moleculesand components of the test fluid that are larger than maltose.

Bias Voltage

In the non-enzymatic glucose-sensing system, the addition ofmaltose-blocking layer 301 does not require an increase or decrease ofthe bias voltage for glucose sensing.

Porous Polymeric Layer

In embodiments, the maltose-blocking layer 301 is made of or includes aporous polymeric material through which glucose may pass but maltose maynot pass. The porous polymeric material contains at least onepoly-phenylenediamine (poly-PD) which include poly(m-phenylenediamine)(poly-mPD), poly(-phenylenediamine) (poly-oPD), andpoly(p-phenylenediamine) (poly-pPD).

Nano-Sized Thickness

The maltose-blocking layer 301 has a thickness at or about 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nm.Throughout the discussions, the thickness of maltose-blocking layerrefers to an average thickness of the polymer layer excluding the top10% and bottom 10% of thickness variations. In embodiments, thethickness may be within a range formed by selecting any two numbers (twothickness values) listed in the immediately previous sentence, e.g.,between about 15 nm and about 35 nm, between about 17 nm and about 33nm, between about 18 nm and about 32 nm, between about 20 nm and about30 nm, between about 21 nm and about 29 nm, between about 22 nm andabout 28 nm, etc.

Level of Porosity

In embodiments, the maltose-blocking layer 301 has porosity that allowsglucose molecules to pass through its thickness while effectivelyblocking maltose molecules from passing therethrough. To accomplish thegoal of allowing glucose to pass and blocking maltose from passing, theoverall porosity of the maltose-blocking layer needs to be adjusted to adesirable level. The overall porosity of the maltose-blocking layer 301relate to the density (or internal morphology including pores andchannels) and the thickness of the layer. The concentration of amaterial for the maltose-blocking layer and method of forming themaltose-blocking layer may be relevant to the density. While there hasbeen some successes of adjusting the overall porosity using theseparameters, it has been found that the level of porosity may not begenerally defined or described using the concentration of the materialand method of forming the layer. While thickness of the maltose-blockinglayer also relates to the overall porosity, it is dependent upon thespecific porosity or porosity per volume. Thus, the level of porosityneeds to be defined in a different manner.

Sensitivity (Current Density) for Glucose and Maltose withoutMaltose-Blocking Layer

For glucose monitoring, at steady state with the application of a biasvoltage of 0.2-0.45 V in a test fluid with the glucose concentration of4-20 mM (typical glucose level in human bodily fluid), the nanoporouslayer 117 contacting the test fluid (i.e., no maltose-blocking layer)needs to generate glucose-oxidation current (electric current caused byoxidation of glucose alone) at a level higher than 10 nA/mMcm², theminimum current density (sensitivity) for glucose. According toembodiments, without the maltose-blocking layer the same nanoporouslayer 117 would generate a similar level of electric current (i.e.,higher than 10 nA/mMcm²) at steady state with the application of a biasvoltage of 0.2-0.45 V in a test fluid containing maltose at aconcentration of 4-20 mM (the same as glucose concentration as above).

Porosity of Maltose-Blocking Layer by Current Density of Glucose andMaltose

According to embodiments, the maltose blocking layer 301 has porosityfor allowing glucose to travel therethrough such that the glucoseoxidation current is still higher than the minimum current density forglucose. Accordingly, when applying a bias voltage of 0.2-0.45 V in atest fluid with the glucose concentration of 4-20 mM, at steady statethe working electrode 103NE with the maltose blocking layer 301generates glucose-oxidation current at a level higher than 10 nA/mMcm²,the minimum current density (sensitivity) for glucose. On the otherhand, the maltose blocking layer 301 has porosity that effectively blockmaltose from passing therethrough such that, when applying a biasvoltage of 0.2-0.45 V in a test fluid with the maltose concentration of4-20 mM, at steady state electric current caused by maltose alone(maltose-oxidation current) is at a level lower than 5 nA/mMcm², themaximum current density for maltose with the maltose-blocking layer.

Electrochemical Polymerization

The porous polymer material for the maltose-blocking layer 301 may beformed on the nanoporous layer 117 by electrochemical polymerization(electropolymerization) using a cyclic voltammetric technique. Inembodiments, a working electrode including the nanoporous layer issubmerged in a reaction mixture solution containing monomer for thecyclic voltammetric electrochemical polymerization. By applying a biasvoltage between the working and reference electrodes within the range ofthe monomer's oxidation voltage, polymerization occurs and a polymerlayer is formed on the nanoporous layer. More details for thepolymerization of phenylenediamine are disclosed in“Electropolymerization of O-Phenylenediamine on Pt-Electrode fromAqueous Acidic Solution: Kinetic, Mechanism, Electrochemical Studies andCharacterization of the Polymer Obtained”, Sayyah et al, Journal ofApplied Polymer Science, Vol. 112, Issue 6, 3695-3706 (2009), and“Electropolymerization of P-Phenylenediamine on Pt-Electrode fromAqueous Acidic Solution: Kinetics, Mechanism, Electrochemical Studies,and Characterization of the Polymer Obtained”, Sayyah et al, Journal ofApplied Polymer Science, Vol. 117, Issue 2, 943-952 (2010), each ofwhich is hereby incorporated herein by reference.

Application of Oxidation Voltage

The bias voltage may be varied during the cyclic voltammetry. Forexample, the bias voltage may be gradually increased within theoxidation voltage range for the initial time segment and then graduallydecreased within the oxidation voltage range for the following timesegment, although not limited thereto. For phenylenediamine, the biasvoltage is applied between 0.5 V and 1.0 V. FIG. 22 illustrates anexample of scanning the bias voltage during the cyclic voltammetricelectrochemical polymerization of phenylenediamine.

Bias Voltage Scanning Speed

Together with the concentration of monomer discussed below, the scanningspeed of the bias voltage between the lower end and the upper end of theoxidation voltage range may be relevant to the porosity and thickness ofthe resulting polymer layer. In embodiments, the scanning speed is atabout 0.5, 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90,100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 350 or 400mV/sec. In embodiments, the scanning speed may be within a range formedby selecting any two numbers listed in the immediately previoussentence, e.g., between about 5 mV/sec and about 200 mV/sec.

Concentration of Monomer

The concentration of the monomer is at about 0.01, 0.05, 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4,2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2,5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0,8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8 or 10 mM. In embodiments,the concentration of the monomer may be within a range formed byselecting any two numbers listed in the immediately previous sentence,e.g., between about 0.05 mM and about 0.8 mM, between about 1.0 mM andabout 5.0 mM, etc. The foregoing concentrations are applicable to thethree species of phenylenediamine.

Porosity in View of Monomer Concentration

The concentration of monomer in the reaction mixture solution isrelevant to the porosity of the resulting maltose-blocking layer. In theflowchart for making the maltose-blocking layer of FIG. 24, the monomerconcentration is determined first at step 2401 and polymerization iscarried out at step 2403. In embodiments, the monomer concentrationunder about 0.7 mM, about 0.6 mM, or about 0.5 mM may provide adesirable level of overall porosity for the maltose-blocking layer. Inembodiments, when the monomer concentration is over about 0.7 mM, about0.8 mM, about 0.9 mM, about 1.0 mM, about 1.1 mM, or about 1.2 mM, theresulting polymer layer does not have enough porosity to allow glucoseto pass therethrough, i.e., generating glucose-oxidation current at alevel lower than 10 nA/mMcm², the minimum current density (sensitivity)for glucose. At step 2405, the resulting polymer layer is subject totreatment for adjusting its porosity at step 2405.

Electric Shock for Adjusting Porosity

When the overall porosity of the polymer layer 302 is not at a desirablelevel, the polymer layer may be further treated for adjusting theporosity. For example, the polymer layer may be subject to an electricshock. In embodiments, the electric shock may be applied to the polymerlayer 302 using the chronoamperometry setting illustrated in FIG. 23, inwhich an electric shock electrode 309 and polymer layer 302 formed onthe nanoporous layer 117 are submerged in an electrolyte solution 311. Avoltage supply 305 and a switch 307 are connected between the substrate303 and the electric shock electrode 309. With the operation of theswitch 307, electric current flows through the porous polymer layer 302and causes morphology changes, which increases porosity of the polymerlayer 302. As a result, the polymer layer 302 turns to themaltose-blocking layer 301 having a desirable level of porosity thatallows glucose to pass through its thickness and effectively blockmaltose from passing therethrough.

Acidic Solution

The electrolyte solution for the electric shock may be an acidicsolution having pH under about 2, 3 or 4 although not limited thereto.In some embodiments, the acidic solution may contain at least one acid.Non-limiting examples of acid for the acidic solution include phosphoricacid (H₃PO₄), nitric acid (HNO₃), chloric acid (HCl), formic acid,lactic acid, malic acid, citric acid, carbonic acid, sulfonic acid, etc.

Waveform for Electric Shock

The electric potential may be applied in various waveforms. Inembodiments, the electric potential is applied in AC or DC. Inembodiments, the electric potential is applied in multiple pulses or ina single pulse. In embodiments, the electric potential may be applied inother shapes of voltage signals.

Electric Potential for Electric Shock

The electric potential applied to the polymer layer 302 is about at orabout 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0,3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 V. In embodiments,the maximum voltage may be within a range formed by selecting any twonumbers listed in the immediately previous sentence, e.g., between about0.5 and about 2.5 V, between about 1.0 and about 2.0 V, etc.

Period for Electric Shock

The period of applying electric potential is for or about 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3,3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4 or 4.5 seconds. Inembodiments, the period may be within a range formed by selecting anytwo numbers listed in the immediately previous sentence, e.g., betweenabout 0.5 and about 2.5 sec., between about 1.0 and about 2.0 sec., etc.

Maltose-Blocking Layer Also Applicable to Enzymatic Sensing

In embodiments, the maltose-blocking layer 301 may be applied toenzymatic glucose-sensing systems. Referring back to FIG. 2, themaltose-blocking layer 301 may be added as an additional functionallayer 112 over the enzyme layer 111 to block maltose while lettingglucose pass therethrough.

CGM Working Electrode CGM System

A continuous glucose monitoring (CGM) system includes a glucose-sensingelectrode that contacts biological fluid of a subject in vivo formeasurement of glucose level contained in the biological fluid. Inpractice, a CGM electrode is inserted or implanted in the subject's bodyfor measurement over an extended period, such as a few days, a week,weeks or months.

Non-enzymatic CGM Working Electrode

FIG. 31 illustrates a cross-section of a non-enzymatic CGM workingelectrode 501 according to an embodiment. The illustrated CGM workingelectrode 501 has a laminated structure that includes a base 503, aconductive layer 110, a nanoporous layer 117, a maltose-blocking layer301, an electrolyte ion-blocking layer 505, and a biocompatibility layer507.

Electrode Base

The base, base substrate or electrode base 503 provides a support forthe laminated structure of the CGM working electrode 501. Inembodiments, the base 503 is an electrically insulative layer and may bemade of or contain a material such as, but not limited to, polyimide,polypropylene, polyethylene glycol, polyhydroxyethyl methacrylate(pHEMA) and other biocompatible polymers. In embodiments, the base 503may be in the form of a flexible film of an electrically insulating andbiocompatible material. The base 503 has a thickness ranging betweenabout 30 μm and about 200 μm, although not limited thereto. The base 503is an optional layer for the CMG sensing electrode 501 and may beomitted in some embodiments.

Conductive Layer

The conductive layer 110 may be placed over the base 503 with or withoutan intervening layer therebetween. In embodiments, the conductive layer110 is formed by printing or dispensing a conductive or semiconductivematerial on the base 503, although not limited thereto. In the CGMworking electrode 501, the conductive layer 110 may have a thicknessranging between about 100 nm and 100 μm, although not limited thereto.In some embodiments, the conductive layer 119 may include two or moresublayers of conductive or semiconductive materials. In embodimentswhere the base 503 is omitted, the conductive layer 119 may function asa support for the laminated structure over it.

Nanoporous Layer

The nanoporous layer 117 may be formed on the conductive layer 110. Inthe CGM working electrode 501, the nanoporous layer 117 has a thicknessranging between about 500 nm and about 10 μm, although not limitedthereto. The nanoporous layer 117 may have at least one of clusteredmorphology, non-clustered morphology, hexagonal nanostructure or othernanoporous morphology.

Maltose-Blocking Layer

The maltose-blocking layer 301 may be formed on the nanoporous layer 117to block maltose molecules from reaching the underlying nanoporous layer117 while allowing glucose molecules to pass therethrough. Inembodiments, the maltose-blocking layer 301 includes a polymericmaterial such as poly-PD having nano-sized pores for passing glucosemolecules and not passing maltose molecules. The maltose-blocking layermay have a thickness ranging between about 5 nm and about 40 nm,although not limited thereto. The maltose-blocking layer 301 is anoptional layer for the CMG sensing electrode 501 and may be omitted insome embodiments.

Electrolyte Ion-Blocking Layer (Electrode ConditioningEnhancement/Facilitation Layer)

The electrolyte ion-blocking layer 505 effectively limits or inhibitssmall electrolyte ions such as Na⁺, K⁺, Ca²⁺, Cl⁻, PO₄ ³⁻ and CO₃ ²⁻from passing therethrough or diffusing toward the underlying nanoporouslayer 117. As will be discussed later, the electrolyte ion-blockinglayer 505 enhances conditioning of the CGM working electrode and alsoreferred to as a working electrode conditioning enhancement orfacilitation layer. The electrolyte ion-blocking layer 505 is porous sothat glucose molecules can freely pass therethrough. When implemented,the electrolyte ion-blocking layer 505 is hydrophobic such that it wouldnot quickly swell by absorbing water contained in the test fluid. Theelectrolyte ion-blocking layer 505 may have a thickness ranging betweenabout 0.1 μm and about 10 μm, although not limited thereto.

Materials for Electrolyte Ion-Blocking Layer

The electrolyte ion-blocking layer 505 may include or be made of atleast one of, for example, poly(methyl methacrylate) (PRIMA),poly(hydroxyethyl methacrylate) (PHEMA), and poly(methylmethacrylate-co-ethylene glycol dimetbactylate) (PMMA-EG-PMMA). Also,the electrolyte ion-blocking layer 505 may be formed of or additionallyinclude a copolymer of methylmethacrylate and butylmethacrylate, andpolymers obtained from polymerization of one or more monomers includingmethylmethacrylate, ethylmethacrylate, propylmethacrylate,butylmethacrylate, pentylmethacrylate, hexylmethacrylate,cyclohexylmethacrylate, 2-ethylhexylmethacrylate, methylacrylate,ethylacrylate, propylacrylate, butylacrylate, pentylacrylate,hexylacrylate, cyclohexylacrylate, 2-ethylhexylacrylate.

Biocompatibility Layer

The biocompatibility or bioprotection layer 507 interfaces with tissuesand bodily fluid of the subject when the CGM sensor is implanted orinserted in the subject's body. The biocompatibility layer 507 containsat least one biocompatible material that is not toxic to the tissues ofthe subject and does not cause immunological rejection by the subject'sbody. Also, the at least one material of the biocompatibility layer 507should allow bodily fluid to pass therethrough to reach the underlyingnanoporous layer 117 such that sensing of glucose concentration is notsignificantly compromised by its own existence. The biocompatibilitylayer 507 may have a thickness ranging between about 5 μM and about 30although not limited thereto.

Materials for Biocompatibility Layer

The biocompatibility layer 507 may include or be made of at least oneof, for example, poly(vinylalcohol),poly(ethyleneoxide-copropyleneoxide) (PEO-PPO), poly(ethyleneoxide)(PEO), poly(sulphone) (PS), poly(ethylene terephthalate) (PET),poly(ether-urethanes) (PU), poly(dimethylsiloxane) (PDMS),ethylene-co-vinylacetate (EVA), poly(methylmethacrylate),poly(tetrafluoroethylene) (PTFE), poly(propylene) (PP), poly(ethylene)(PE), polyethylene glycol, and polyhydroxyethyl methacrylate (pHEMA).

Modifications

The CGM working electrode 501 may include one or more additionalfunctional layers although not shown in FIG. 31. In some embodiments,one or more of the maltose-blocking layer 301, electrolyte ion-blockinglayer 505 and biocompatibility layer 507 may be omitted. In otherembodiments, two or more of the maltose-blocking layer 301, electrolyteion blocking layer 505 and biocompatibility layer 507 may be combined inone layer or change their locations.

No Enzyme Layer

The CGM working electrode 501 does not include an enzyme layercontaining a glucose-specific enzyme. Nor does the CGM working electrode501 contain any such enzyme in any of the layers.

No Oxygen Take-Up Layer

The CGM working electrode 501 does not include an oxygen take-upmaterial or layer that would be needed for collecting and supplyingmolecular oxygen in case glucose oxidase is used for oxidation ofglucose.

No Electron Mediator

The CGM working electrode 501 does not include an electron mediationmaterial that would be needed for transferring electrons in case glucosedehydrogenase is used for oxidation of glucose. Conditioning CGM WorkingElectrode or System

Transient Signals of Electric Current

Upon creating an electrochemical cell using a CGM working electrode withthe application of a bias voltage, the CGM working electrode generateselectric current. The electric current from the CGM working electroderepresents the sum of background noises and electric current fromglucose oxidation in the CGM working electrode. Initially, the electriccurrent shows a transient behavior. As shown in FIGS. 25-30, in thebeginning the electric current is very high compared to that caused byglucose oxidation alone and rapidly decreases. Subsequently, the rate ofdecrease slows down. Ultimately, the electric current settles at alevel, i.e., steady state, although in vivo the current may fluctuate abit within a tolerable range.

Electric Current for Glucose Sensing

For accurate glucose sensing, the electric current should be measuredwhen the electrochemical cell and/or CGM working electrode are in asteady state. In other words, the electric current from a CGM workingelectrode should not change too much over time (i.e., settling at alevel after the initial decrease) when the glucose concentration doesnot change. Further, for accurate glucose sensing, the backgroundcurrent (noises) should not be too high relative to the electric currentcaused by glucose oxidation alone. In other words, the total electriccurrent should not be too high relative to the electric current fromglucose oxidation alone.

Conditioning CGM Working Electrode or Electrochemical Cell

CGM working electrodes need conditioning before glucose sensing. Here,conditioning refers to the process of stabilizing CGM working electrodesfor accurate glucose sensing. Upon completion of conditioning of a CGMworking electrode, the electric current therefrom should settle at alevel and should not be too high relative to the electric current fromglucose. To provide accurate glucose level, a CGM system should useelectric current measured after conditioning is finished. Conditioningof a CGM working electrode may take a long time. Commercially availableenzymatic CGM working electrodes requires several hours to days forconditioning.

Desirable Rate of Electric Current Change

Given that the electric current from glucose oxidation in vivo is abouttens of nano Ampere, for accurate glucose sensing, the decrease rate ofthe electric current from a CGM working electrode should be smallerthan, for example, 20 nA (nano Ampere) per minute. For the sake ofproviding a reference point, the desirable rate of the electric currentchange should be a point at or below 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 nA per minute. In embodiments, the rateof electric current change may be determined in a shorter or longer timeperiod. s

Desirable Level of Electric Current

Electric current from glucose oxidation in vivo is typically tens ofnano Ampere. The desirable level of total electric current may changedepending upon various factors including measurement accuracy, signalprocessing capability, data processing capability, etc. As these factorsare further developed, the desirable level could increase. Nonetheless,given that the electric current from glucose oxidation in vivo is abouttens of nano Ampere, for accurate glucose sensing, the electric currentfrom a CGM working electrode should be smaller than, for example, 500nA. For the sake of providing a reference point, the desirable electriccurrent should be a point at or below 500, 490, 480, 470, 460, 450, 440,430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300,290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160,150, 140, 130, 120, 110 or 100 nA.

Completion of Conditioning

A CGM system determines that conditioning of its CGM working electrodeor its electrochemical cell is complete. The CGM system may determinecompletion of conditioning when the rate of electric current change isor stays at or below a predetermined value, e.g., a desirable rate ofelectric current change or decrease as set forth above. The CGM systemdetermines completion of conditioning when the total electric currentchange stays for a predetermined time at or below a predetermined value,e.g., a desirable level of electric current as set forth above. The CGMsystem may determine completion of conditioning when the rate ofelectric current change is or stays at or below its predetermined valueand further when the total electric current change stays for apredetermined time at or below its predetermined value, e.g., the rateof electric current change being less than 5 nA/min and the totalelectric current staying less than 400 nA for 1 minute.

Notifying Completion of Conditioning

A CGM system may notify its user of completion of conditioning. Upon orsometime after forming the electrochemical cell for glucose oxidation,the CGM system may begin monitoring the electric current from its CGMworking electrode. When the electric current meets one or morerequirements for the completion of conditioning, the CGM system mayprovide a notification to its user for notifying the completion ofconditioning. The notification may be in any form including sound,vibration, light or information display. In addition or in thealternative, the CGM system may not provide any information indicating aglucose level prior to completion of conditioning.

Reducing Time for Conditioning CGM Working Electrode

Concentration Discontinuity of Small Electrolyte Ions Human bodily fluidcontains a significant amount of electrolyte ions of Na⁺, K⁺, Ca²⁺, Cl⁻,PO₄ ³⁻ and CO₃ ²⁻. In embodiments, the electrolyte ion-blocking layer505 limits or inhibits the electrolyte ions of Na⁺, K⁺, Ca²⁺, Cl⁻, PO₄³⁻ and CO₃ ²⁻ from passing therethrough. As a result, between above theelectrolyte ion-blocking layer 505 and below the same layer, theconcentration of these electrolyte ions are significantly different.FIG. 32 conceptually illustrates the concentration discontinuity on bothsides of the electrolyte ion-blocking layer 505. With the electrolyteion-blocking layer 505, the combined concentration of the smallelectrolyte ions are significantly smaller in the nanoporous layer 117than in the biocompatibility layer 507. Without the electrolyteion-blocking layer 505, the combined concentration of the smallelectrolyte ions in the nanoporous layer 117 would be similar to that inthe biocompatibility layer 507.Concentration of Small Electrolyte Ions under Electrolyte Ion-BlockingLayer

In embodiments, the combined concentration of the electrolyte ions belowthe electrolyte ion-blocking layer 505 is greater than 0% but lower thanabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or 20% of the combined concentration of the same electrolyte ions abovethe electrolyte ion-blocking layer 505. The combined concentration belowthe electrolyte ion-blocking layer 505 may be within a range formed byselecting any two numbers (two % values) listed in the immediatelyprevious sentence. As illustrated in FIG. 32, for example, the combinedconcentration of the electrolyte ions in human interstitial bodily fluid(i.e., above the electrolyte ion-blocking layer 505) is about 0.1 M orhigher; in contrast, the combined concentration of the electrolyte ionsbelow the electrolyte ion-blocking layer 505 is about 0.01 M or lower.The combined concentration of the electrolyte ions below the electrolyteion-blocking layer 505 may be obtained by measuring double-layercapacitance of the nanoporous layer 117 and applying the measured valueto the Gouy-Chapman formula, as discussed in detail in IonicStrength-Controlled Virtual Area of Mesoporous Platinum Electrode, Booet al, J. AM. CHEM. Soc. 2004, 126, 4524-4525.

Acceleration of Ionic Equilibrium in Nanoporous Layer

As discussed, the ion-blocking layer 505 establishes or creates asubstantial discontinuity in the combined concentration of the smallelectrolyte ions between over the electrolyte ion-blocking layer 505 andunder the same layer. The low concentration of the small electrolyteions significantly excels conditioning of the CGM working electrode 501,particularly conditioning of the nanoporous layer 117. Although anyaspects of the invention are not bound by any theory or belief, the lowconcentration of the small electrolyte ions may accelerate ionicequilibrium in nano-sized structures and surfaces of the nanoporouslayer 117 that would not occur in larger scale such as micro-sizedstructures and surfaces. As the ionic equilibrium is accelerated in thenanoporous layer 117, the time for reaching ionic equilibrium or steadystate inside the nano-structures of the nanoporous layer 117 would beshorter at a lower concentration of the electrolyte ions with theexistence of the electrolyte ion-blocking layer 505 than at a higherconcentration without the electrolyte ion-blocking layer 505.

Significantly Shorter Time for Conditioning

With the acceleration of ionic equilibrium in the nanoporous layer 117,the electrolyte ion-blocking layer 505 significantly enhances andfacilitates conditioning of the non-enzymatic CGM working electrode 501of FIG. 31, i.e., shortening the time for reaching a desirable electriccurrent and/or a desirable rate of electric current change, i.e., steadystate. According to embodiments, a fraction of time is needed for thecompletion of conditioning when using a non-enzymatic CGM workingelectrode 505 with the electrolyte ion-blocking layer 505 compared towhen using the same non-enzymatic CGM working electrodes without anelectrolyte ion-blocking layer 505.

Conditioning Time

When the desirable rate of electric current change is 5 nA/min or less,a non-enzymatic CGM working electrode without an electrolyteion-blocking layer 505 take about 3 hours in serum that containselectrolyte ions at 0.1 M or higher; in contrast, a non-enzymatic CGMworking electrode with an electrolyte ion-blocking layer 505 takes lessthan at or about 1 hour and 30 minutes, 1 hour and 25 minutes, 1 hourand 20 minutes, 1 hour and 15 minutes, 1 hour and 10 minutes, 1 hour and5 minutes, 1 hour, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35minutes, or 30 minutes in the same serum. When the desirable rate ofelectric current change is 3 nA/min or less, a non-enzymatic CGM workingelectrode without an electrolyte ion-blocking layer 505 take more than 5hours in serum that contains electrolyte ions at 0.1 M or higher; incontrast, a non-enzymatic CGM working electrode with an electrolyteion-blocking layer 505 takes less than at or about 1 hour and 30minutes, 1 hour and 25 minutes, 1 hour and 20 minutes, 1 hour and 15minutes, 1 hour and 10 minutes, 1 hour and 5 minutes, 1 hour, 55minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25minutes, 15 minutes or 10 minutes in the same serum. When the desirablerate of electric current change is 2 nA/min or less, a non-enzymatic CGMworking electrode without an electrolyte ion-blocking layer 505 takemore than 5 hours or 10 hours in serum that contains electrolyte ions at0.1 M or higher; in contrast, a non-enzymatic CGM working electrode withan electrolyte ion-blocking layer 505 takes less than at or about 1 hourand 30 minutes, 1 hour and 25 minutes, 1 hour and 20 minutes, 1 hour and15 minutes, 1 hour and 10 minutes, 1 hour and 5 minutes, 1 hour, 55minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25minutes, 15 minutes or 10 minutes in the same serum.

Unexpected Results

Without proper conditioning, a CGM working electrode may not provideelectric current for an accurate glucose level. Reducing the time forconditioning is a very important practical consideration in developingand manufacturing a CGM working electrode. This is because properconditioning of a CGM working electrode may take hours, if not tens ofminutes, and because there is a tendency that people would want to knowtheir glucose level immediately after inserting the electrode in theirbody. Referring to examples discussed later, the time for conditioningCGM working electrode is reduced from about 3, 5 or 10 hours to lessthan 30 minutes by including an electrolyte ion-blocking layer 505 alonewith all the other conditions being the same. This is very significantimprovement and unexpectedly high achievement.

Specifics of Electrolyte Ion-Blocking Layer

The electrolyte ion-blocking layer 505 of a non-enzymatic CGM workingelectrode includes or is made of at least one porous, hydrophobicpolymer including poly(methyl methacrylate) (PMMA), poly(hydroxyethylmethacylate) (PHEMA), and poly(methyl methacrylate-co-ethylene glycoldimethacrylate) (PMMA-EG-PMMA). Additional examples of the porous,hydrophobic polymer include a copolymer of methylmethacrylate andbutylmethacrylate, and polymers obtained from polymerization of one ormore monomers including methylmethacrylate, ethylmethacrylate,propylmethacrylate, butylmethacrylate, pentylmethacrylate,hexylmethacrylate, cyclohexylmethacrylate, 2-ethylhexylmethacrylate,ethylacrylate, ethylacrylate, propylacrylate, butylacrylate,pentylacrylate, hexylacrylate, cyclohexylacrylate, 2-ethylhexylacrylate,etc. The average molecular weight for these polymers is about 5,000,10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000,100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000,180,000, 190,000, 200,000, 210,000, 220,000, 230,000, 240,000, 250,000,260,000, 270,000, 280,000, 290,000, 300,000, 310,000, 320,000, 330,000,340,000, 350,000, 360,000, 370,000, 380,000, 390,000 or 400,000. Inembodiments, the molecular weight may be within a range formed byselecting any two numbers listed in the immediately previous sentence.The electrolyte ion-blocking layer may have a thickness of about 0.1,0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5 or 10 μm. In embodiments, the thickness may be within a range formedby selecting any two numbers (two thickness values) listed in theimmediately previous sentence, e.g., between about 2 and about 5 μm,ranging between about 1 and about 3 μm, etc.

Ion Concentration Drop Has No Effect in Enzymatic Glucose SensingElectrode

In an enzymatic CGM system, a CGM working electrode includes aglucose-specific enzyme for oxidation of glucose molecules. Theenzymatic CGM working electrode may include a functional layercontaining a porous, hydrophobic material that may effectively dropconcentration of electrolyte ions under the functional layer. In theenzymatic CGM system, however, the concentration drop by the functionallayer may not provide a reduction of the time for conditioning of theCGM electrode that relates to ionic equilibrium in nano-sized surfacesor structures. This is because the enzymatic CGM system uses enzymes foroxidizing glucose molecules and does not require a nanoporous layer forglucose oxidation. Accordingly, even if a porous, hydrophobic layer isincluded in an enzymatic CGM working electrode, even if such a layercauses discontinuity of electrolyte ion concentration across itsthickness, and further even if there is some reduction of time forconditioning of the enzymatic CGM working electrode, such reductionwould not be equated to the reduction of time for conditioning in thenon-enzymatic CGM working electrode 501 having both the electrolyte ionblocking layer 505 and nanoporous layer 117.

CGM Subcutaneous Electrode Module CGM Electrode Unit

In embodiments, the CGM system includes an electrode unit or module forsubcutaneously contracting bodily fluid of a subject. The electrode unitmay include a single body accommodating one or more electrodes thatwould contact the bodily fluid when inserted into the subject's body.The single body may be flexible.

Construction of CGM Electrode Unit

FIG. 33 illustrates a CGM electrode unit 701 according to an embodiment.The CGM electrode unit 701 includes a subcutaneous portion 703 and acontact terminal portion 705. The subcutaneous portion 703 is forinserting into the subject's body and includes a working electrode 501,a counter electrode 105 and a reference electrode 106 that are exposedvia openings formed through an insulating layer 707 for subcutaneouslycontacting bodily fluid. The contact terminal portion 705 is for stayingoutside the subject's body and for engaging or connecting with acounterpart device. The contact terminal portion 703 includes a workingelectrode terminal 501T, a counter electrode terminal 105T and areference electrode terminal 106T that are electrically connected to theworking electrode 501, counter electrode 105 and reference electrode106, respectively, underneath the insulation layer 707. Here, each ofthe working electrode 501, counter electrode 105 and reference electrode106 may have features and characteristics as discussed in the presentdisclosure, although not limited thereto. Fabricating CGM Electrode Unit

FIG. 34 is a flowchart for fabricating the CGM electrode unit 701according to an embodiment. At step 3401, an electrically insulative,flexible film is provided for a base or electrode base 503 (also in FIG.31). Subsequently at step 3403, a conductive layer is formed on the base503 in predetermined shapes 110R, 110W and 110C as illustrated in FIG.35. Subsequently as step 3405, an insulation film 707 is applied overthe conductive layer to selectively expose portions or areas of theconductive layer as in FIG. 36. Subsequently at step 3407, theintermediate product is cut to provide the shape as illustrated in FIG.37. At step 3409, a nanoporous layer 117 is formed on an exposed areafor the working electrode 501. Subsequently at 3411, one or morefunctional layers are formed on the nanoporous layer 117 to provide alaminated construction of the non-enzymatic CGM working electrode 501 asin FIG. 31. Further, a salt layer may be formed on the exposed area forthe reference electrode 106. In embodiments, cutting the intermediateproduct at step 3407 may be performed after step 3409 or 3411.

Conductive Layer—Multiple Conductive Elements

FIG. 35 provides a top view of an intermediate product after step 3403according to an embodiment and its cross-section taken along the line3501 and viewed in the arrow direction. As illustrated, the conductivelayer formed on the base 503 has three separate elements 110C, 110W and110R in predetermined shapes, i.e., conductive layer element 110C forcounter electrode, conductive layer element 110W for working electrode,and conductive layer element 110R for reference electrode. Each of theconductive layer elements 110C, 110W and 110R includes a conductiveportion reserved for a contact terminal (in the contact terminal portion705 of FIG. 33), a conductive portion reserved for an electrode (in thesubcutaneous portion 703 of FIG. 33), and a conductive connectionbetween the two conductive portions.

Making Conductive Layer

The conductive layer may be in a single layer of an electricallyconductive material or formed of multiple sublayers of differentconductive materials. In embodiments, either or both of the conductivelayer element 110C for counter electrode and the conductive layerelement 110W for working electrode are formed of at least two sublayers,e.g., a silver layer and a conductive carbon layer over the silverlayer. In embodiments, the conductive layer element 110R for referenceelectrode is formed in a single layer, e.g. a silver layer. Theconductive layer 110 or its sublayers may be formed by printing aconductive ink on or over the base 503 and subsequent drying. A sublayerformed on another sublayer may be also formed by printing a conductivematerial for that sublayer. The conductive layer elements 110W, 110C and110R of FIG. 35 are all in a single layer; for the purpose of showingalternatives, however, in FIGS. 36-38, the conductive layer elements110W and 110C have a two-sublayer construction, i.e., carbon layer 1605over silver layer 1603 (see also FIG. 16A).

Insulation Film

FIG. 36 illustrates an intermediate product after placing the insulationfilm according to an embodiment. The insulation film 707 may be pre-cutwith openings in the subcutaneous portion 703 of FIG. 33 for exposingconductive portions reserved for the counter electrode 105, workingelectrode 501 and reference electrode 106. The insulating film 707 doesnot cover the contact terminal portion 705 of FIG. 33 and accordinglyexposes the terminal portion of each of the conductive layer elements110C, 110W and 110R, which become 105T, 501T and 106T, respectively. Theconductive connections of the conductive layer elements 110C, 110W and110R are covered with the insulation film 707. An adhesive layer (notillustrated) may be interposed between the base film 503 and theinsulation film 707. The insulation film 707 may be an adhesive-coatedfilm.

Cutting

At step 3407, the intermediate product of FIG. 36 is subject to cuttingto remove unnecessary portions of the insulation film 707 and base 503,for example, by die cutting. FIG. 37 illustrates the resulting product,in which the contact terminal portion 705 (proximal end portion of theCGM electrode unit 701) is wider than the subcutaneous portion 703(distal end portion of the CGM electrode unit 701). In embodiments, thedistal portion has a width of about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 mm in the direction alongthe line 3501. In embodiments, the width may be within a range formed byselecting any two numbers listed in the immediately previous sentence,e.g., between about 1.0 mm and about 1.5 mm. In embodiments, the CGMelectrode unit 701 has a length of about 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30mm in a direction between the distal end and proximal end thereof. Inembodiments, the length may be within a range formed by selecting anytwo numbers listed in the immediately previous sentence, e.g., betweenabout 10 mm and about 20 mm.

Forming Nanoporous Layer

At step 3409, a nanoporous layer 117 is formed on the conductive layerelement 110W exposed for working electrode. FIG. 38A illustrates across-section of the intermediate product taken along the line 3501 inthe arrow direction after forming the nanoporous layer 117. Inembodiments, the nanoporous layer 117 is formed by dispensing clustercolloid containing nanoparticle clusters dispersed in liquid over theconductive layer 110 and drying the liquid off therefrom. In thealternative, another form of the nanoporous layer 117 may be formedusing a different method as disclosed herein. In some embodiments,cutting at step 3407 may be performed subsequent to forming thenanoporous layer 117.

Functional Layer(s) for Working Electrode

Subsequent to forming the nanoporous layer 117, one or more functionallayers are formed on the nanoporous layer 117 to provide thenon-enzymatic CGM working electrode 501 as in FIG. 31. A maltoseblocking layer 301 may be formed on the nanoporous layer 117, althoughnot limited thereto. An electrolyte ion-blocking layer 505 may be formedover the nanoporous layer 117 for the improvement in conditioning theresulting CGM working electrode 501, although not limited thereto.Further, a biocompatibility layer 507 may be formed over the nanoporouslayer 117, more specifically over the electrolyte ion-blocking layer505, although not limited thereto. FIG. 38B illustrates a cross-sectionof the CGM working electrode 501 including electrolyte ion-blockinglayer 505 and biocompatibility layer 507.

Reference and Counter Electrodes

In embodiments, a salt layer, e.g., AgCl may be formed on the conductivelayer element 110R exposed for the reference electrode 106. Forming thesalt layer may be performed any time after forming the conductive layerelement 110R. In embodiments, the counter electrode 105 may not requirean additional treatment over the conductive layer element 110C.

Subcutaneous Insertion of CGM Electrode Unit

In embodiments, the subcutaneous portion 703 (distal portion) of the CGMelectrode unit 701 is subcutaneously inserted into the subject's bodywith or without use of an insertion tool that is known in the art orwill be developed in the future. With proper subcutaneous insertion, theworking electrode 501, reference electrode 106 and counter electrode 105of the subcutaneous portion 703 contact the subject's interstitialbodily fluid while the terminal portion 705 of the CGM electrode unit701 stays outside the subject's body.

Counterpart Device

Subsequently, in embodiments, the terminal portion 705 is engaged orconnected with a counterpart device (not illustrated) that includescounterpart ports or terminals corresponding to the working electrodeterminal 501T, counter electrode terminal 105T and reference electrodeterminal 106T. In embodiments, the counterpart device further includesan electrical circuit that completes the electrochemical cell of FIG. 1together with the CGM electrode unit 701 for continuous monitoringglucose module. In some embodiments, in addition to the electricalcircuit for completing an electrochemical cell, the counterpart devicemay include at least one processor for processing data includingelectric current obtained from the electrochemical cell to convert to astandardized number representing a glucose level. In some embodiments,the counterpart device includes a wireless module for wirelessly sendingdata to another wireless device such as a smartphone or computingdevice.

BGM Disposable Strip Single Point in Time Devices

Glucose sensing may be performed in vitro at a single point in time. Asingle-point-in-time glucose-sensing system measures a glucose level ina test fluid, most commonly blood. Accordingly, the system is referredto as a blood glucose monitoring (BGM) system. The BGM systems include asingle-use disposable cartridge or strip.

Disposable Cartridge

FIG. 39 illustrates a BGM disposable cartridge 901 and a sensing module911 for a single-point-in-time glucose-sensing system according toembodiments. The disposable cartridge 901 includes a test fluidreservoir 903, a counter electrode 105, a reference electrode 106, and acartridge working electrode 905 formed on a base 907 that provides astructural support for the electrodes 105, 106 and 905. Electricconnections (not shown) are formed between the electrodes and aconnector 909 through the base 907.

Sensing Module

In embodiments, the disposable cartridge 901 is designed to electricallyand/or mechanically couple with the sensing module 911 via the connector909. The sensing module 911 may include electric circuitry (not shown)for a voltage source 109 and a current sensor 108. When the disposablecartridge 901 is properly connected to the sensing module 911, theelectrodes 105, 106 and 905 are connected to the circuit of the sensingmodule 911 in a manner similar to FIG. 1.

Working Electrode

The working electrode 905 according to an embodiment, which includes aconductive layer 110 and a nanoporous layer 117. The working electrode905 further includes a filter layer 913 to filter and screen cells,lipid and large molecules contained in the test fluid. In embodiments,the filter layer 913 may be made of or include woven cloth, cotton orother materials that can screen cells, lipid and other large componentsof blood while passing glucose therethrough.

Working Electrode Does Not Include

In embodiments, the working electrode 905 contains no glucose-specificenzyme. Further, the working electrode 905 contains no surfactant and noelectron mediator that may be necessary in enzymatic glucose sensing.Further, given that the working electrode 905 is an in vitro device, itdoes not require a biocompatibility layer either.

Calibration of Working Electrode

Electric Current from Working Electrode

According to embodiments, the non-enzymatic working electrode with ananoporous glucose-oxidation layer generates electric current caused byoxidation of glucose contained in a test liquid. In practice, theelectric current from the non-enzymatic working electrode includes 1)electric current caused by glucose oxidation alone (glucose-oxidationcurrent), 2) electric current caused by interfering chemical entities ifthe test fluid contains such, and 3) electric current caused byinteractions between the electrochemical cell and other chemicalentities contained in the test fluid.

Glucose Levels in Bodily Fluid

Normal glucose levels in healthy individuals are between 4.0 and 6.0 mM(between 72 and 108 mg/dL). Considering diabetic patients, the glucoselevels may range between 4.0 and 20 mM (between 72 and 360 mg/dL).

Glucose-Oxidation Current

In embodiments, at steady state (after conditioning) in a test fluidcontaining 4.0-20 mM glucose, when applying a bias voltage between about0.2 V and about 0.45 V, the electric current from glucose oxidationalone (glucose-oxidation current) is at a level higher than 10 nA/mMcm².In the glucose concentration range of 4.0-20 Mm, the nanoporousglucose-oxidation layer (hence, the non-enzymatic working electrode)generates the glucose-oxidation current at about 0.5, 1.0, 1.5, 2.0,2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 or 6.0 nA for 1 mM of glucosecontained in the test fluid. In embodiments, the glucose-oxidationcurrent from 1 mM of glucose contained in the test fluid may be within arange formed by any two numbers in the immediately preceding sentence,e.g., between about 1.5 nA and 2.5 nA. Accordingly, for the glucoseconcentration range of 4.0-20 mM, the glucose-oxidation current from thenon-enzymatic working electrode may be between about 2.0 nA (4.0×0.5)and about 120 nA (20×6.0). In embodiments, the glucose-oxidation currentmay be about 2.0, 4.0, 8.0, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118 or 120 Na. In embodiments,the glucose-oxidation current from 4.0-20 mM glucose contained in thetest fluid may be within a range formed by any two numbers in theimmediately preceding sentence, e.g., between about 1.5 nA and 2.5 nA.

Calibration of Electric Current and Glucose Concentration

In embodiments, for the same glucose concentration in the test fluid,the glucose-oxidation current may differ from one nanoporousglucose-oxidation layer to another, depending upon their particularmanufacturing conditions. Also, in a particular nanoporousglucose-oxidation layer, the glucose-oxidation current is generallylinearly correlated with the glucose concentration, although it may notbe so linear throughout the concentration or electric current range. Inembodiments, for each batch of nanoporous glucose-oxidation layersmanufactured using the same of conditions, one or more nanoporousglucose-oxidation layers are tested to determine the correlation profilebetween glucose-oxidation current and glucose concentration for theparticular batch. Later in the process of glucose sensing or monitoringusing a nanoporous glucose-oxidation layer from the same batch, thecorrelation profile is used in computing or determining a glucose levelin a test fluid.

Second Working Electrode Ascorbic Acid

Ascorbic acid is known as Vitamin C and plays an important role in thehuman body. Ascorbic acid is prone to oxidation and is readily oxidizedat a low oxidation potential. Ascorbic acid may interfere with glucosesensing from bodily fluid.

Currently No Layer Available for Blocking Ascorbic Acid

Given that ascorbic acid is negatively charged, a negatively chargedlayer has been proposed to repel ascorbic acid while passing glucose.However, no glucose-sensing electrode is commercially available to blockascorbic acid so far.

Two Working Electrodes

In embodiments, a glucose sensor or sensing system includes at least oneadditional working electrode in addition to the working electrode 103 ofFIG. 1. FIG. 40 conceptually illustrates a two-working electrodeglucose-sensing system 4101. In this system, a first working electrode4103A, a second working electrode 4103B, a counter electrode 105 and areference electrode 106 are connected to a potentiostat 4104, whichincludes electric circuitry for functioning as op-amps 4107A and 4107B,current sensors 4108A and 4108B, and voltage sources 4109A and 4109B forthe two working electrodes 4103A and 4103B.

Operation of Two-Working Electrode System

In embodiments, oxidation of both glucose and ascorbic acid occurs atthe first working electrode 4103A. Accordingly, electric current fromthe first working electrode 4103A represents the combined concentrationof glucose and ascorbic acid in the test fluid 102. On the other hand,at the second working electrode 4103B, oxidation of the ascorbic acidoccurs but oxidation of glucose does not occur. Accordingly, electriccurrent from the second working electrode 4103B represents only theconcentration of ascorbic acid in the same test fluid 102. Thedifference between the two electric current values represents theconcentration or level of glucose contained in the test fluid 102.

First Working Electrode (Glucose Working Electrode)

In some embodiments, the first working electrode (glucose workingelectrode) 4103A includes the nanoporous layer 117 over the conductivelayer 110, as in FIG. 3. The nanoporous layer 117 may include clusterednanoporous structure, although not limited thereto. In otherembodiments, the first working electrode 4103A may include an enzymelayer containing glucose-specific enzyme for oxidizing glucose, as inFIG. 2, instead of the nanoporous layer 117 of FIG. 3. In eitherembodiment, the first working electrode 4103A does not include anegatively charged membrane or any other membrane for inhibitingascorbic acid from passing therethrough.

Second Working Electrode (No-Glucose Working Electrode)

The second working electrode (no-glucose working electrode) 4103Bincludes a conductive layer 110 but does not include any layers orfeatures for effectively causing oxidation of glucose. In embodiments,the second working electrode 4103B includes neither the nanoporous layer117 nor a glucose-specific enzyme for oxidizing glucose. However,oxidation of ascorbic acid occurs at the conductive layer 110. Inembodiments, the conductive layer 110 includes a conductive carbon layerformed on a silver layer, although not limited thereto.

The Same Bias Voltage for Two Electrodes

In embodiments, the same bias voltage is applied to both the first andsecond working electrodes 4103A and 4103B relative to the referenceelectrode 106. This is to provide an environment to cause about the samelevel of oxidation of ascorbic acid to occur at both the first andsecond working electrodes 4103A and 4103B. Assuming the same level ofoxidation occurs for ascorbic acid at each of the first and secondworking electrodes 4103A and 4103B, the difference between electriccurrent from the first working electrode 4103A and electric current fromthe second working electrode 4103B should represent the oxidation ofglucose at the first working electrode 4103A.

Addressing Interference of Additional Chemical Entities

The two-electrode system 4101 can be used to address the interference ofmore than one chemical entity. In embodiments, by adjusting the biasvoltage, the first working electrode 4103A may oxidize not only glucoseand ascorbic acid but also an additional interfering chemical entitysuch as acetaminophen. Likewise, the second working electrode 4103Boxidizes not only ascorbic acid but also the additional interferingchemical entity at the same time. Here, neither of the first and secondworking electrodes includes any membrane for inhibiting the additionalinterfering chemical entity. Then, the electric current from the firstworking electrode 4103A represents oxidation of glucose, ascorbic acidand acetaminophen, and the electric current from the second workingelectrode 4103B represents oxidation of ascorbic acid and acetaminophen.The difference between the electric currents represents oxidation ofglucose, cancelling off the interference of acetaminophen and ascorbicacid.

Bias Voltage

In embodiments, any bias voltage value within the range of 0.2-0.45 Vmay be used for addressing the interference. In some embodiments, a biasvoltage value within the range of 0.2-0.32 V may be used for addressingthe interference of ascorbic acid alone given that acetaminophen may notbe oxidized in the nanoporous metal layer at that bias voltage range asdiscussed in more detail below.

Different Bias Voltages

In embodiments, the two-electrode system 4101 may adopt different biasvoltages for the first and second working electrodes. For example, afirst bias voltage is applied to the first working electrode 4103A, anda second bias voltage is applied to the second working electrode 4103B.With the different bias voltages, the electric current from oxidation ofascorbic acid at the second working electrode 4103B may not be the sameor equivalent to the current component by oxidation of ascorbic acid atthe first working electrode 4103A. Thus, the current from glucoseoxidation may not be the simple difference between the currents from thetwo electrodes. In embodiments, however, the two-electrode system 4101has or is connected to hardware and software for computing an accurateglucose concentration using the different bias voltages, the currentvalues from the first and second working electrodes 4103A and 4103B,data indicative of oxidation potential of ascorbic acid at the differentbias voltages, etc.

Concomitant Detections

In some embodiments, detection of the current from the first workingelectrode 4103A and detection of the current from the second workingelectrode 4103B occur at the same time, simultaneously, concurrently orconcomitantly. In other embodiments, either with one current sensor ortwo current sensors, the detections may occur at different times with atime gap as long as the concentration fluctuation of the concernedchemical entities is negligible over the time gap. Skilled artisans inthe art would appreciate how long the time gap can be without too muchof the risk of being inaccurate. For example, the time gap is less than1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 seconds, or the time gap is less than 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes.

Recording Concentration of Interfering Chemical

In embodiments, the two-electrode system 4101 includes or is to beconnected to hardware and software (not shown) that is configured tostore current values from the first and second working electrodes 4103Aand 4103B and/or to store respective concentrations of glucose andascorbic acid obtained from the current values. In some embodimentswhere oxidation of both ascorbic acid and acetaminophen occurs at thesecond working electrode 4103B, the hardware and software is configuredto store the concentration of glucose and a combined concentration ofascorbic acid and acetaminophen.

Applicable to CGM

The two-electrode system 4101 may be implemented in a CGM electrode unitfor in vivo glucose sensing. FIG. 41 illustrates a CGM electrode unit4201 including the first and second working electrodes 4103A and 4103B,which are connected to first and second working electrode terminals4103AT and 4103BT respectively.

Applicable to BGM

The two-electrode system 4101 may be implemented in a BGM disposablecartridge or strip for in vitro glucose sensing. In embodiments, thedisposable cartridge 901 of FIG. 39 may include two working electrodes.In such embodiments, the cartridge working electrode 905 works as thefirst working electrode 4103A. The second working electrode 4103B may beadded to the base 907 for contacting the test fluid. Further, thecorresponding sensing module 911 may include circuitry for receivingsignals from the first and second working electrodes from the BGMdisposable cartridge. s

First and Second Working Electrodes Must Operate Together

In the two-electrode system 4101, there must be two current values: onefrom the first working electrode 4103A and the other from the secondworking electrode 4103B in order to obtain a glucose level in the testfluid. For CGM, each of the first and second working electrodes 4103Aand 4103B must operate continuously or repeatedly to provide glucoselevels. Accordingly, the system is distinguished from anyelectrochemical sensing systems having a spare sensing electrode that isoccasionally used for various reasons.

Interference By Acetaminophen Acetaminophen

Acetaminophen is one of the most commonly used over-the-countermedications. Further, acetaminophen is widely used in combinationaldrugs as an active pharmaceutical ingredient.

Well-Recognized Problem

Given the popularity of acetaminophen, it is possible that the drug canbe taken by patients who also need to detect their blood glucose level.Considering that many glucose sensing devices are used by patientsthemselves, not by healthcare professionals, incorrect readings causedby acetaminophen can lead to serious consequences. The industry forelectrochemical glucose sensing has known this problem and beeninterested in solving it.

No Good Solution

There have been many attempts to solve this problem. Thus far, however,no solution has convinced the industry to adopt. No membrane has beenadopted to selectively screen acetaminophen from reaching the electrode.Thus, there is a long-felt-but-unmet need.

Explanation for No Good Solution

The commercially available electrochemical glucose sensing technologiessimply cannot address this issue at all. This is at least in partbecause electrochemical glucose-sensing systems are technically verycomplex. The working electrode has laminated components, each of whichhas its own function and does not interfere with the other components.It would be difficult to find a solution addressing this probleminvolving acetaminophen without affecting the functions of othercomponents and overall performance of the working electrode. In additionto the technical complexity, developing a product like this for marketlaunching is very expensive in view of the rigorous regulatory approvalprocess in this industry. Accordingly, once a working product has beenapproved and launched in the market, significant changes to any workingcomponent of the approved product would be difficult to make.

Non-enzymatic Glucose-Sensing System Addressing Acetaminophen

In embodiments, a non-enzymatic electrochemical glucose-sensing systemselectively oxidizes glucose and at the same time does not oxidizeacetaminophen without introducing any additional membrane for thisresult. Referring back to FIGS. 3 and 31, the working electrode 103NE,501 includes the conductive layer 110 and the nanoporous layer 117. Theworking electrode may include one or more additional functional layersover the nanoporous layer 117.

No Acetaminophen Screening Membrane

In embodiments, the working electrode 103NE does not include, over thenanoporous layer 117, a membrane, film or layer that is designed toselectively screen, repel or block acetaminophen while allowing glucoseto pass therethrough. Thus, when the working electrode 103NE contactsthe test fluid containing acetaminophen, both glucose and acetaminophenwill contact the nanoporous layer 117 and will be able to enternano-sized pores for oxidation therein.

Bias Voltages for Oxidation of Glucose and Acetaminophen

In the glucose-sensing system according to embodiments, glucose isoxidized in the nanoporous layer 117 at a bias voltage between about 0.2V and about 0.45 V. On the other hand, acetaminophen is oxidized at abias voltage greater than 0.33, 0.34, 0.35 or 0.36 V. The bias voltagemay be adjusted to cause oxidation of glucose and to avoid oxidation ofacetaminophen at the same time.

Bias Voltage for Selective Oxidation of Glucose and No Oxidation ofAcetaminophen

In embodiments, the bias voltage applied to the conductive layer 110relative to the reference electrode 106 is set to cause oxidation ofglucose but not to cause oxidation of acetaminophen when both contactthe nanoporous layer 117. For selective oxidation of glucose andselective non-oxidation of acetaminophen, in embodiments, the biasvoltage is set at or about 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26,0.27, 0.28, 0.29, 0.30, 0.31 or 0.32 V. In embodiments, the bias voltagemay be within a range formed by selecting any two numbers (two voltagevalues) listed in the immediately previous sentence, e.g., between 0.28V and 0.30 V, between about 0.27 V and about 0.31 V, between 0.26 V and0.30 V, between about 0.28 V and about 0.32 V, etc. In embodiments, thebias voltage is lower than 0.30, 0.31 or 0.32 V.

Bias Voltage in Enzymatic Sensing Electrode

For the sake of contrast, enzymatic glucose sensors apply the biasvoltage in the range of 0.5-0.6 V. In the enzymatic sensing sensors,this bias voltage does not cause oxidation of glucose at its sensingelectrode or elsewhere. Rather, glucose-specific enzymes oxidize glucosemolecules, which generates electron to an electron mediator that isoxidized at the conductive layer by the bias voltage. Thus, the biasvoltage is to cause oxidation of the electron mediator in the enzymaticelectrode.

EXAMPLES

Now various aspects and features of the invention are further discussedin connection with examples and experiments.

Preparing Reverse Micelle Phase Example 1.1

A platinum aqueous solution was prepared by dissolving 0.500 g (0.965mmol) of chloroplatinic acid hexahydrate H₂PtCl₆.6H₂O (fromSigma-Aldrich) in 24.5 g of purified water with stirring. 25 g ofsurfactant, Triton X-100™ (from Sigma-Aldrich) was added to the platinumaqueous solution to provide an aqueous composition containing surfactantand platinum ions. The concentration of the platinum ions in the aqueouscomposition was about 0.02 M. A reverse micelles phase was prepared inthe aqueous composition by adjusting the temperature to 70° withstirring.

Example 1.2

A reverse micelle phase is prepared by repeating Example 1.1 except thatPtCl₄.6H₂O is used, in replacement of H₂PtCl₆.6H₂O, in an amount toprovide the platinum ion concentration about 0.02 M in the aqueouscomposition.

Example 1.3

A reverse micelle phase is prepared by repeating Example 1.1 except thatH₂PtCl₂(OH)₄ is used, in replacement of H₂PtCl₆.6H₂O, in an amount toprovide the platinum ion concentration about 0.02 M in the aqueouscomposition.

Example 1.4

A reverse micelle phase is prepared by repeating Example 1.1 except thatH₂Pt(SO₄)(OH)₄.6H₂O is used, in replacement of H₂PtCl₆.6H₂O, in anamount to provide the platinum ion concentration about 0.02 M in theaqueous composition.

Example 1.5

A reverse micelle phase is prepared by repeating Example 1.1 except thatTiCl₄.6H₂O is used, in replacement of H₂PtCl₆.6H₂O, in an amount toprovide the titanium ion concentration about 0.02 M in the aqueouscomposition.

Example 1.6

A reverse micelle phase is prepared by repeating Example 1.1 except thatNP-40™ is used as surfactant, in replacement of Triton X-100, to providethe platinum ion concentration about 0.02 M in the aqueous compositionand further except that the amount of surfactant and the temperature areadjusted to achieve a reverse micelle phase of the surfactant.

Example 1.7

A reverse micelle phase is prepared by repeating Example 1.1 except thatpolysorbate 80 is used as surfactant, in replacement of Triton X-100, toprovide the platinum ion concentration about 0.02 M in the aqueouscomposition and further except that the amount of surfactant and thetemperature are adjusted to achieve a reverse micelle phase of theparticular surfactant.

Example 1.8

A reverse micelle phase is prepared by repeating Example 1.1 except thatisoceteth-20 is used as surfactant, in replacement of Triton X-100, toprovide the platinum ion concentration about 0.02 M in the aqueouscomposition and further except that the amount of surfactant and thetemperature are adjusted to achieve a reverse micelle phase of theparticular surfactant.

Example 1.9

A reverse micelle phase is prepared by repeating Example 1.1 except thatpoloxamer 407 is used as surfactant, in replacement of Triton X-100, toprovide the platinum ion concentration about 0.02 M in the aqueouscomposition and further except that the amount of surfactant and thetemperature are adjusted to achieve a reverse micelle phase of theparticular surfactant.

Example 1.10

A reverse micelle phase is prepared by repeating Example 1.1 except thatmonolaurin is used as surfactant, in replacement of Triton X-100, toprovide the platinum ion concentration about 0.02 M in the aqueouscomposition and further except that the amount of surfactant and thetemperature are adjusted to achieve a reverse micelle phase of theparticular surfactant.

Preparing Reducing Agent Example 2.1

A reducing agent aqueous solution was prepared by adding 30 g (0.170mol) of ascorbic acid as a reducing agent to 250 ml of purified waterwith stirring. The reducing agent solution was heated to 70° C. Theconcentration of the ascorbic acid in the reducing agent aqueoussolution was 0.6 M, which is equivalent to 60 times the concentration ofmetal ions of Examples 1.1 through 1.10.

Example 2.2

A reducing agent aqueous solution was prepared by repeating Example 2.1except that form aldehyde is used as reducing agent in replacement ofascorbic acid. The amount of form aldehyde is adjusted to provide itsconcentration in the reducing agent aqueous solution about 0.6 M.

Example 2.3

A reducing agent aqueous solution was prepared by repeating Example 2.1except that acetic acid is used as reducing agent in replacement ofascorbic acid. The amount of acetic acid is adjusted to provide itsconcentration in the reducing agent aqueous solution about 0.6 M.

Example 2.4

A reducing agent aqueous solution was prepared by repeating Example 2.1except that hypophosphite is used as reducing agent in replacement ofascorbic acid. The amount of hypophosphite is adjusted to provide itsconcentration in the reducing agent aqueous solution about 0.6 M.

Forming Nanoparticle Colloid Example 3.1

The reducing agent aqueous solution prepared in Example 2.1 was added tothe aqueous composition of Example 1.1 at 70° C. soon after the reversemicelle phase was prepared. In the resulting liquid composition, theconcentration of platinum ions was about 0.0028 M, and the concentrationof ascorbic acid was about 0.50 M. The resulting liquid composition wascontinuously stirred for about 4 hours at 70° C. A black platinumcolloid was obtained.

Examples 3.2-3.10

Example 3.1 is repeated using the reverse micelle phases prepared inExamples 1.2-1.10, in replacement of the reverse micelle phase preparedin Example 1.1, which provides metal colloids of Examples 3.2-3.10,respectively.

Particle Size Analysis of Nanoparticle Colloid Example 4.1

Korea Polymer Testing and Research Institute (KOPTRI) performed adynamic light-scattering particle size analysis for the platinum colloidobtained from Example 3.1 using Zeta-potential & particle size analyzerELS-Z2 of Photal Otsuka Electronics. For the analysis, a sample of theExample 3.1 platinum colloid was dispersed in purified water havingrefractive index of 1.3328, viscosity of 0.8878 cp, and dielectricconstant of 78.3 at 25° C.

FIG. 14 shows particle size distribution for the colloid obtained fromExample 3.1. The particle diameters are primarily between about 9 nm andabout 14 nm. This size distribution is interpreted as representing thereverse micelles. The size distribution does not show diameter sizes of1-5 nm, which is interpreted as most platinum nanoparticles beingcontained or encompassed inside the reverse micelles. Similar resultswere obtained from multiple runs of the experiments according toExamples 1.1, 2.1 and 3.1.

Examples 4.2-4.10

The analysis of Example 4.1 is repeated using each colloid prepared inExamples 3.2-3.10, in replacement of the colloid prepared in Example3.1. A particle size distribution for each of the colloid prepared inExamples 3.2-3.10 is obtained.

Removing Surfactant Example 5.1

50 ml of 0.3 M HCl aqueous solution was added to 60 ml of the platinumcolloid prepared in Example 3.1. The acid-added platinum colloid wascentrifuged for 10 minutes at 3800 rpm. Subsequently, clear supernatantwas discarded, and black bottom portion was collected. The sequence ofadding HCl aqueous solution, centrifugation and collecting black bottomportion was repeated four additional times to remove the surfactant andobtain a platinum colloid.

Subsequently, the resulting platinum colloid was washed with purifiedwater to remove HCl. 50 ml of purified water was added to the collectedplatinum colloid. The water-added platinum colloid was centrifuged for10 minutes at 3800 rpm. Then, clear supernatant was discarded, and blackbottom portion was collected. The sequence of adding purified water,centrifugation and collecting black bottom portion was repeated fouradditional times to remove HCl and obtain an HCl-washed platinumcolloid.

Examples 5.2-5.10

Example 5.1 is repeated using the nanoparticle colloid obtained fromExamples 3.2-3.10, in replacement of the nanoparticle colloid preparedin Example 3.1 to collect colloids of Examples 5.2-5.10 respectively.

Example 5.11

Example 5.1 is repeated using 0.3 M HNO₃ aqueous solution in replacementof HCl aqueous solution.

Example 5.12

Example 5.1 is repeated using 0.3 M NaOH aqueous solution in replacementof HCl aqueous solution.

Particle Size Analysis of Cluster Colloid Example 6.1

Korea Polymer Testing and Research Institute (KOPTRI) performed adynamic light-scattering particle size analysis for the platinum colloidobtained from Example 5.1 using Zeta-potential & particle size analyzerELS-Z2 of Photal Otsuka Electronics as in Example 4.1. For the analysis,a sample of the Example 5.1 colloid was dispersed in water havingrefractive index of 1.3328, viscosity of 0.8878 cp, and dielectricconstant of 78.3 at 25° C.

FIG. 15 shows particle size distribution for the colloid obtained fromExample 5.1. The particle diameters are primarily between about 60 nmand about 200 nm. This size distribution is interpreted as representingirregularly shaped clusters formed of nanoparticles. Considering thatthe particle size in Example 4.1 is primarily between about 9 nm andabout 14 nm (the size of a reverse micelle and not of clusters), it isunderstood that clusters were formed by the processes of Example 5.1, inwhich surfactant molecules were detached from platinum nanoparticles byaddition of acidic solution and the surfactant was removed bycentrifugation and collection of bottom portion. Similar results wereobtained from multiple runs of the experiments according to Examples1.1, 2.1, 3.1 and 5.1.

Examples 6.2-6.10

Example 6.1 is repeated using each colloid prepared in Examples3.2-3.10, in replacement of the colloid prepared in Example 3.1. Aparticle size distribution for each colloid prepared in Examples3.2-3.10 is obtained.

Recovery of Platinum—Yield Example 7

The cluster colloid obtained in Example 5.1 was subjected to drying. Thedry weight of the colloid was 0.143 g. The resulting colloid in Example5.1 was prepared from 60 ml of the nanoparticle colloid prepared inExample 3.1, which contained 0.188 g. In the overall process, the yieldof platinum was 76.1%.

Making Electrode with Clustered Nanoporous Layer Example 8.1 ElectrodeBase

A silver layer 1603 and a conductive carbon layer 1605 were formed on asubstrate 1601 made of polyimide as illustrated in FIG. 16A. The silverlayer 1603 was formed in a thickness of about 20 μm by printing a silverink containing silver particles. The conductive carbon layer 1605 wasformed in a thickness of about 20 μm by printing a carbon ink containingcarbon particles. A polyimide insulation film 1602 was laminated overthe substrate 1601 around the silver layer 1603 and conductive carbonlayer 1605 to provide the electrode base 1606.

Example 8.2 Forming Nanoporous Layer

The cluster colloid obtained in Example 5.1 was diluted to theconcentration of 60 mg/ml. Using a micro-syringe, 0.2 μL of the dilutedcluster colloid was dropped on the conductive carbon layer of theelectrode base 1606. The electrode base with the colloid dropped thereonwas placed in an oven at 60° C. for 30 minutes to form an electrode 1607including a platinum nanoporous layer 1609 as illustrated in FIG. 16B.

Example 8.3 Roughness Factor

The electrochemical cell of FIG. 1 was prepared using electrochemicalanalyzer CHI660 from CH Instruments Inc. as potentiostat 104 and usingthe electrode 1607 prepared in Example 8.2 as working electrode 103, aplatinum wire as counter electrode 105, and Ag/AgCl (3 M KCl) asreference electrode 106. The silver layer 1603 of the electrode 1607 wasconnected to the potentiostat 104. Instead of the test fluid 102, 1MH₂SO₄ aqueous solution was added to the electrochemical cell of FIG. 1.

Cyclic voltammetry was performed with potential sweeping between −0.2 Vand +1.2 V. The real surface area of the platinum nanoporous layer wasobtained by measuring the amount of proton adsorbed to the surfaces ofplatinum nanoporous layer using the cyclic voltammetry. The top surfacearea (geometric area) of the platinum nanoporous layer was measured.Roughness factor was calculated by dividing the real surface area by thegeometric area. The roughness factor of the nanoporous layer obtainedfrom Example 8.2 was 1147.

Example 8.4 Repeating Examples 8.1-8.2

Examples 8.1 was repeated multiple times to prepare additional electrodebases. Example 8.2 was repeated multiple times using the additionalelectrode bases to prepare additional electrodes 1607 including aplatinum nanoporous layer 1609.

Example 8.5 Repeating Example 8.3

Example 8.3 was repeated for the five electrodes 1607 prepared inExample 8.4. The roughness factor values of the nanoporous layers were1187, 1171, 1143, 1190 and 1119.

Example 8.6 SEM Photographs

FIG. 17A is an SEM photograph taken from the top of an electrode 1607obtained from Example 8.4. The darker center represents the area of theconductive carbon layer. FIG. 17B is an SEM photograph of across-section of the electrode 1607 showing the platinum nanoporouslayer 1609, carbon conductive layer 1605 and silver layer 1603 insequence from the top. FIG. 17C includes three SEM photographs ofanother electrode 1607 prepared in Example 8.4. These three photographsare taken from the top in different magnifications.

Sensing Glucose in PBS Example 9.1 Preparing Solutions of Glucose andOther Test Materials

D-(+)-glucose powder purchased from Sigma-Aldrich was dissolved inpurified water to prepare 1 M glucose stock solution. Ascorbic acidpurchased from Sigma-Alddrich was dissolved in purified water to prepare0.05 M ascorbic Sigma-Aldrich acid aqueous solution. Acetaminophenpurchased from Sigma-Aldrich was dissolved in purified water to prepare0.05 M acetaminophen aqueous solution. Maltose purchased fromSigma-Aldrich was dissolved in purified water to prepare 0.5 M maltoseaqueous solution.

Example 9.2 Preparing PBS

500 ml aqueous solution containing 0.1 M NaH₂PO₄ and 0.15 M NaCl inpurified water was prepared. 500 ml aqueous solution containing 0.1 MNa₂HPO₄ and 0.15 M NaCl in purified water was prepared. The two aqueoussolutions were mixed to prepare 1 L stock phosphate buffered saline(PBS) in pH 7.4.

Example 9.3 Preparing Glucose-Sensing System in PBS

20 ml of the PBS prepared in Example 9.2 was placed in a beaker, inwhich the temperature of PBS was maintained at 37° C. Theelectrochemical cell of FIG. 1 was prepared using electrochemicalanalyzer CHI660 from CH Instruments Inc. as potentiostat 104 and usingthe electrode 1607 prepared in Example 8.4 as working electrode 103, aplatinum wire as counter electrode 105, and Ag/AgCl (3 M KCl) asreference electrode 106. The silver layer 1603 of the electrode 1607 wasconnected to the potentiostat 104. The electrodes were submerged intoPBS and electrically connected to the electrochemical analyzer.

Example 9.4 Measuring Electric Current

In the system prepared in Example 9.3, the bias voltage of 0.4 V wasapplied between the working electrode 103 (electrode 1607) and thereference electrode 106. Upon application of the bias voltage, electriccurrent from the working electrode 103 was continuously measured. Theelectrochemical cell was kept for 12 minutes for conditioning theglucose-sensing system in PBS without addition of any substance thereto.Subsequently, the current value of 0.087 μA was taken for no glucosecontained in the PBS. FIG. 18 shows the electric current profileobtained from the electrochemical cell for Examples 9.5-9.11 below. InFIG. 18, “AA” represents ascorbic acid, and “AP” representsacetaminophen.

Example 9.5 Sensing 1 mM Glucose in PBS

After conditioning of the glucose-sensing system, 20 μl of the glucosestock solution prepared in Example 9.1 was added to the PBS of Example9.3 to make 1 mM glucose in the PBS. Immediately after the addition, theglucose-added PBS was stirred for 3-4 seconds, which caused temporarypeaks of electric current. The electric current from the workingelectrode was continuously measured. When the current became stable, thecurrent value of 0.54 μA was taken for 1 mM glucose in PBS.

Example 9.6 Sensing 3 mM Glucose in PBS

After the current became stable in Example 9.5, 40 μl of the glucosestock solution prepared in Example 9.1 was added to the PBS resultingfrom Example 9.4 to make the total glucose 3 mM in the PBS. Immediatelyafter the addition, the glucose-added PBS was stirred for 3-4, seconds,which caused temporary peaks of electric current. The electric currentfrom the working electrode was continuously measured. When the currentbecame stable, the current value of 1.19 μA was taken for 3 mM glucosein PBS.

Example 9.7 Sensing 6 mM Glucose in PBS

After the current became stable in Example 9.6, 60 μl of the glucosestock solution prepared in Example 9.1 was added to the PBS resultingfrom Example 9.5 to make the total glucose 6 mM in the PBS. Immediatelyafter the addition, the glucose-added PBS was stirred for 3-4, seconds,which caused temporary peaks of electric current. The electric currentfrom the working electrode was continuously measured. When the currentbecame stable, the current value of 2.09 μA was taken for 6 mM glucosein PBS.

Example 9.8 Sensing 10 mM Glucose in PBS

After the current became stable in Example 9.7, 80 μl of the glucosestock solution prepared in Example 9.1 was added to the PBS resultingfrom Example 9.6 to make the total glucose 10 mM in the PBS. Immediatelyafter the addition, the glucose-added PBS was stirred for 3-4, seconds,which caused temporary peaks of electric current. The electric currentfrom the working electrode was continuously measured. When the currentbecame stable, the current value of 2.89 μA was taken for 10 mM glucosein PBS.

Example 9.9 Sensing 0.11 mM Ascorbic Acid in PBS

After the current became stable in Example 9.8, 44 μl of the ascorbicacid aqueous solution prepared in Example 9.1 was added to the PBSresulting from Example 9.7 to make 0.11 mM ascorbic acid (AA) in thePBS. Immediately after the addition, the ascorbic acid-added PBS wasstirred for 3-4, seconds, which caused temporary peaks of electriccurrent. The electric current from the working electrode wascontinuously measured. When the current became stable, the current valueof 2.93 μA was taken for the sum of 10 mM glucose and 0.11 mM ascorbicacid in PBS.

Example 9.10 Sensing 0.17 mM Acetaminophen in PBS

After the current became stable in Example 9.9, 68 μl of theacetaminophen aqueous solution prepared in Example 9.1 was added to thePBS resulting from Example 9.8 to make 0.17 mM acetaminophen (AP) in thePBS. Immediately after the addition, the acetaminophen-added PBS wasstirred for 3-4, seconds, which caused temporary peaks of electriccurrent. The electric current from the working electrode wascontinuously measured. When the current became stable, the current valueof 3.21 μA was taken for the sum of 10 mM glucose, 0.11 mM ascorbic acidand 0.17 mM acetaminophen in PBS.

Example 9.11 Sensing 13.9 mM Maltose in PBS

After the current became stable in Example 9.10, 556 μl of the maltoseaqueous solution prepared in Example 9.1 was added to the PBS resultingfrom Example 9.9 to make 13.9 mM maltose in the PBS. Immediately afterthe addition, the maltose-added PBS was stirred for 3-4, seconds, whichcaused temporary peaks of electric current. The electric current fromthe working electrode was continuously measured. When the current becamestable, the current value of 4.74 μA was taken for the sum of 10 mMglucose, 0.11 mM ascorbic acid, 0.17 mM acetaminophen and 13.9 mMmaltose in PBS.

Example 9.12 Glucose Level Formula

In Examples 9.5-9.11, the current values represent and correspond to theconcentrations of glucose in the PBS. Similar experiments are conductedmany more times for the glucose-sensing system prepared in the samemanner using the same and other glucose concentrations to obtain data ofcurrent values and corresponding glucose concentrations. A correlationbetween glucose concentration and current value in the PBS is obtainedby processing the data. Glucose concentrations are computed using thecorrelation and the current values obtained from Examples 9.5-9.11.

Sensing Glucose in Serum Example 10.1 Preparing Glucose-Sensing Systemin Serum

Human serum was purchased from Sigma-Aldrich. The glucose content in theserum was measured using YSI. It was determined that the serum contained5.8 mM glucose therein, which corresponds to blood glucose level 104mg/dl. 10 ml of the serum was placed in a beaker, in which thetemperature of the serum was maintained at 37° C. An electrochemicalcell was prepared as in Example 9.3 except that one electrode 1607prepared in Example 8.4 was used as working electrode 103 and furtherexcept that the working, reference and counter electrodes were submergedinto the serum.

Example 10.2 Pre-Conditioning Glucose-Sensing System in Serum

0.4 V bias voltage was applied between the working electrode 103 and thereference electrode 106 of the electrochemical cell prepared in Example10.1. The bias voltage was maintained for over 3 hours in theelectrochemical system for conditioning the system, i.e., waiting forthe background current to become low enough for sensing glucoseoxidation. Subsequently, the bias voltage was disconnected from thesystem.

Example 10.3 Measuring Electric Current

Soon after removing the bias voltage in Example 10.2, the same biasvoltage was reapplied to the system, and measuring of electric currentfrom the working electrode began. The electrochemical cell was kept for1.2 hours for further conditioning the glucose-sensing system in theserum without adding any substance thereto. When the current becamestable, the current value of 96 nA was taken for 5.8 mM glucoseoriginally contained in serum. FIG. 19 shows a profile of the electriccurrent measured from the electrochemical cell of Examples 10.4-10.9below. In FIG. 19, “AA” represents ascorbic acid, and “AP” representsacetaminophen.

Example 10.4 Sensing 10 mM Glucose in Serum

After conditioning of the glucose-sensing system, 42 μl of the glucosestock solution prepared in Example 9.1 was added to the serum of Example10.2 to make the total glucose 10 mM in the serum. Immediately after theaddition, the glucose-added serum was stirred for 3-4, seconds, whichcaused temporary peaks of electric current. The electric current fromthe working electrode was continuously measured. When the current becamestable, the current value of 110 nA was taken for 10 mM glucose inserum.

Example 10.5 Sensing 15 mM Glucose in Serum

After the current became stable in Example 10.4, 50 μl of the glucosestock solution prepared in Example 9.1 was added to the serum of Example10.3 to make the total glucose 15 mM in the serum. Immediately after theaddition, the glucose-added serum was stirred for 3-4, seconds, whichcaused temporary peaks of electric current. The electric current fromthe working electrode was continuously measured. When the current becamestable, the current value of 132 nA was taken for 15 mM glucose inserum.

Example 10.6 Sensing 20 mM Glucose in Serum

After the current became stable in Example 10.5, 50 μl of the glucosestock solution prepared in Example 9.1 was added to the serum of Example10.4 to make the total glucose 20 mM in the serum. Immediately after theaddition, the glucose-added serum was stirred for 3-4, seconds, whichcaused temporary peaks of electric current. The electric current fromthe working electrode was continuously measured. When the current becamestable, the current value of 159 nA was taken for 20 mM glucose inserum.

Example 10.7 Sensing 0.11 mM Ascorbic Acid in Serum

After the current became stable in Example 10.6, 22 μl of the ascorbicacid aqueous solution prepared in Example 9.1 was added to the serumresulting from Example 10.5 to make 0.11 mM ascorbic acid (AA) in theserum. Immediately after the addition, the ascorbic acid-added serum wasstirred for 3-4, seconds, which caused temporary peaks of electriccurrent. The electric current from the working electrode wascontinuously measured. When the current became stable, the current valueof 163 nA was taken for the sum of 20 mM glucose and 0.11 mM ascorbicacid in serum.

Example 10.8 Sensing 0.17 mM Acetaminophen in Serum

After the current became stable in Example 10.7, 34 μl of theacetaminophen aqueous solution prepared in Example 9.1 was added to theserum resulting from Example 10.6 to make 0.17 mM acetaminophen (AP) inthe serum. Immediately after the addition, the acetaminophen-added serumwas stirred for 3-4, seconds, which caused temporary peaks of electriccurrent. The electric current from the working electrode wascontinuously measured. When the current became stable, the current valueof 223 nA was taken for the sum of 20 mM glucose, 0.11 mM ascorbic acidand 0.17 mM acetaminophen in serum.

Example 10.9 Sensing 13.9 mM Maltose in Serum

After the current became stable in Example 10.8, 278 μl of the maltoseaqueous solution prepared in Example 9.1 was added to the serumresulting from Example 10.7 to make 13.9 mM maltose in the serum.Immediately after the addition, the maltose-added serum was stirred for3-4, seconds, which caused temporary peaks of electric current. Theelectric current from the working electrode was continuously measured.When the current became stable, the current value of 231 nA was takenfor the sum of 20 mM glucose, 0.11 mM ascorbic acid, 0.17 mMacetaminophen and 13.9 mM maltose in serum.

Example 10.10 Glucose Level Formula

In Examples 10.4-10.9, the current values represent and correspond tothe concentrations of glucose in the serum. Similar experiments areconducted many more times for the glucose-sensing system prepared in thesame manner using the same and other glucose concentrations to obtaindata of current values and corresponding glucose concentrations. Acorrelation between glucose concentration and current value in the serumis obtained by processing the data. Glucose concentrations are computedusing the correlation and the current values obtained from Examples10.4-10.9.

Non-Clustered Nanoporous Layers Example 11.1 Electroplating from ReverseMicelle Phase

This disclosure hereby incorporates herein the examples and discussionsof the U.S. Pat. No. 8,343,690 ('690 patent) in its entirety. Theexperiments appearing at columns 6 through 9 of the '690 patent arespecifically incorporated herein as examples for making nanoporous layerby electroplating and using the layer for glucose sensing.

Example 11.2 Electroplating from Hexagonal Phase

This disclosure hereby incorporates herein the disclosure of U.S. Pat.No. 7,892,415 ('415 patent) in its entirety. The experiments appearingat columns 5 through 6 of the '415 patent are specifically incorporatedherein as examples for making hexagonal structured nanoporous layer byelectroplating and using the layer for glucose sensing.

Example 11.3 Electroplating from Hexagonal Phase

This disclosure hereby incorporates herein the disclosure of“Electrochemistry Communications, Vol. 4, Issue 8, August 2002, pages610-612” in its entirety.

Example 11.4 Chemical Deposition from Hexagonal Phase

This disclosure hereby incorporates herein the disclosure of “Science,Vol. 278, Oct. 31, 1997, pages 838-840” in its entirety.

Making Maltose-Blocking Layer Example 12.1 Preparing Aqueous mPDSolutions

M-phenylenediamine (mPD) purchased from Sigma-Aldrich was dissolved inpurified water to provide aqueous mPD solutions containing mPD in 0.1,0.3, 0.5, 1.0, 2.0 and 5.0 mM.

Example 12.2 Preparing for Cyclic Voltammetry

An electrochemical cell was prepared using electrochemical analyzer CHIMulti 1030C from CH Instruments Inc. as potentiostat 104 and using theelectrode 1607 prepared in Example 8.4 as working electrode 103, aplatinum wire as counter electrode 105, and Ag/AgCl (3 M KCl) asreference electrode 106. The counter electrode 105 and referenceelectrode 106 were electrically connected to form a two-electrodesystem.

Example 12.3 Electrochemical Polymerization at 0.1 mM, 10 mV/sec

In the electrochemical cell prepared in Example 12.2, the 0.1 mM aqueousmPD solution prepared in Example 12.1 was added instead of the testfluid 102. Cyclic voltammetry was performed with potential sweepingbetween +0.5 V and +1.0 V at the scanning rate of 10 mV/sec for twosweeping segments as illustrated in FIG. 22, which resulted in apoly-mPD maltose-blocking layer 301 on the nanoporous layer 117.

Example 12.4 Electrochemical Polymerization at 0.1 mM, 100 mV/sec

Example 12.3 was repeated except that the scanning rate was 100 mV/sec,which formed a poly-mPD layer on the nanoporous layer 117.

Example 12.5 Electrochemical Polymerization at 0.1 mM, 200 mV/sec

Example 12.3 was repeated except that the scanning rate was 200 mV/sec,which formed a poly-mPD layer on the nanoporous layer 117.

Example 12.6 Electrochemical Polymerization at 0.3 mM, 10 mV/sec

Example 12.3 was repeated except that the 0.3 mM aqueous mPD solutionprepared in Example 12.1 was added instead of the 0.1 mM aqueous mPDsolution, which formed a poly-mPD layer on the nanoporous layer 117.

Example 12.7 Electrochemical Polymerization at 0.3 mM, 100 mV/sec

Example 12.6 was repeated except that the scanning rate was 100 mV/sec,which formed a poly-mPD layer on the nanoporous layer 117.

Example 12.8 Electrochemical Polymerization at 0.3 mM, 200 mV/sec

Example 12.6 was repeated except that the scanning rate was 200 mV/sec,which formed a poly-mPD layer on the nanoporous layer 117.

Example 12.9 Electrochemical Polymerization at 0.5 mM, 10 mV/sec

Example 12.3 was repeated except that the 0.5 mM aqueous mPD solutionprepared in Example 12.1 was added instead of the 0.1 mM aqueous mPDsolution, which formed a poly-mPD layer on the nanoporous layer 117.

Example 12.10 Electrochemical Polymerization at 0.5 mM, 100 mV/sec

Example 12.6 was repeated except that the scanning rate was 100 mV/sec,which formed a poly-mPD layer on the nanoporous layer 117.

Example 12.11 Electrochemical Polymerization at 0.5 mM, 200 mV/sec

Example 12.6 was repeated except that the scanning rate was 200 mV/sec,which formed a poly-mPD layer on the nanoporous layer 117.

Example 12.12 Electrochemical Polymerization at 1.0 mM, 10 mV/sec

Example 12.3 was repeated except that the 1.0 mM aqueous mPD solutionprepared in Example 12.1 was added instead of the 0.1 mM aqueous mPDsolution, which formed a poly-mPD layer on the nanoporous layer 117.

Example 12.13 Electric Shock

The electrochemical cell of FIG. 23 was prepared for chronoamperometryusing the poly-mPD layer prepared in Example 12.12 as the porous polymerlayer 302 and 1 M H₂SO ₄ aqueous solution as the electrolyte solution.Electric shock was applied to the porous polymer layer 302 by applying asingle pulse from +0.0 V to +1.0 with the pulse width of 1.0 sec.

Example 12.14 Electrochemical Polymerization at 1.0 mM, 100 mV/sec andElectric Shock

Example 12.6 was repeated except that the scanning rate was 100 mV/sec,which formed a poly-mPD layer on the nanoporous layer 117. Subsequently,Example 12.13 was repeated using the poly-mPD layer formed on thenanoporous layer.

Example 12.15 Electrochemical Polymerization at 1.0 mM, 200 mV/sec andElectric Shock

Example 12.6 was repeated except that the scanning rate was 200 mV/sec,which formed a poly-mPD layer on the nanoporous layer 117. Subsequently,Example 12.13 was repeated using the poly-mPD layer formed on thenanoporous layer.

Example 12.16 Electrochemical Polymerization at 2.0 mM, 10 mV/sec andElectric Shock

Example 12.3 was repeated except that the 2.0 mM aqueous mPD solutionprepared in Example 12.1 was added instead of the 0.1 mM aqueous mPDsolution, which formed a poly-mPD layer on the nanoporous layer 117.Subsequently, Example 12.13 was repeated using the poly-mPD layer formedon the nanoporous layer.

Example 12.17 Electrochemical Polymerization at 2.0 mM, 100 mV/sec andElectric Shock

Example 12.6 was repeated except that the scanning rate was 100 mV/sec,which formed a poly-mPD layer on the nanoporous layer 117. Subsequently,Example 12.13 was repeated using the poly-mPD layer formed on thenanoporous layer.

Example 12.18 Electrochemical Polymerization at 2.0 mM, 200 mV/sec andElectric Shock

Example 12.6 was repeated except that the scanning rate was 200 mV/sec,which formed a poly-mPD layer on the nanoporous layer 117. Subsequently,Example 12.13 was repeated using the poly-mPD layer formed on thenanoporous layer.

Example 12.19 Electrochemical Polymerization at 5.0 mM, 10 mV/sec andElectric Shock

Example 12.3 was repeated except that the 5.0 mM aqueous mPD solutionprepared in Example 12.1 was added instead of the 0.1 mM aqueous mPDsolution, which formed a poly-mPD layer on the nanoporous layer 117.Subsequently, Example 12.13 was repeated using the poly-mPD layer formedon the nanoporous layer.

Example 12.20 Electrochemical Polymerization at 5.0 mM, 100 mV/sec andElectric Shock

Example 12.6 was repeated except that the scanning rate was 100 mV/sec,which formed a poly-mPD layer on the nanoporous layer 117. Subsequently,Example 12.13 was repeated using the poly-mPD layer formed on thenanoporous layer.

Example 12.21 Electrochemical Polymerization at 5.0 mM, 200 mV/sec andElectric Shock

Example 12.6 was repeated except that the scanning rate was 200 mV/sec,which formed a poly-mPD layer on the nanoporous layer 117. Subsequently,Example 12.13 was repeated using the poly-mPD layer formed on thenanoporous layer.

Sensing Glucose without Interference by Maltose Example 13.1 PreparingSerum

Human serum was purchased from Sigma-Aldrich. The glucose content in theserum was measured using YSI. It was determined that the serum contained5.8 mM glucose therein, which corresponds to blood glucose level 104mg/dl.

Example 13.2 Preparing Glucose-Sensing System in Serum

10 ml of the serum prepared in Example 13.1 was placed in a beaker, inwhich the temperature of the serum was maintained at 37° C. Anelectrochemical cell was prepared as in Example 10.2 except that theworking electrode 103 includes a poly-mPD maltose-blocking layer 301 onnanoporous layer as prepared in Example 12.3 using 0.1 mM mPD solutionand scanning rate of 10 mV/sec.

Example 13.1 Preparing Glucose-Sensing System in Serum

An electrochemical cell was prepared by repeating Example 10.2 exceptthat the working electrode 103 includes a poly-mPD maltose-blockinglayer 301 on nanoporous layer as prepared in Example 12.3 (using 0.1 mMmPD solution and scanning rate of 10 mV/sec) and further except that theworking, reference and counter electrodes were submerged into the serum.

Example 13.2 Conditioning Glucose-Sensing System in Serum

In the electrochemical cell system prepared in Example 13.1, biasvoltage 0.4 V was applied between the working electrode 103 and thereference electrode 106. The bias voltage was maintained for over 3hours in the electrochemical system for pre-conditioning the system.Subsequently, the bias voltage was disconnected from the system andreconnected. Upon re-application of the bias voltage, measuring ofelectric current from the working electrode began. The electrochemicalcell was kept for further conditioning the glucose-sensing system in theserum. When the current became stable, the current value of 96 nA wasmeasured for 5.8 mM glucose originally contained in serum.

Example 13.3 Electrode with Maltose-Blocking Layer (0.1 mM, 10 mV/sec)

In the system prepared in Example 13.2, the glucose stock solutionprepared as in Example 9.1 was added to the serum to make the totalglucose concentration 10 mM in the serum. Subsequently, the glucosestock solution added further to make the total glucose concentration 15mM and 20 mM in the serum with a time interval between the additions.Subsequently, the ascorbic acid aqueous solution prepared in Example 9.1was added to the serum to make 0.11 mM ascorbic acid in the serum.Subsequently, the acetaminophen aqueous solution prepared in Example 9.1was added to the resulting serum to make 0.17 mM acetaminophen in theserum. Further subsequently, the maltose aqueous solution prepared inExample 9.1 was added to the resulting serum to make 13.9 mM maltose inthe serum. Immediately after each addition, the serum was stirred for3-4 seconds, which caused temporary peaks of electric current. FIG. 25shows the electric current monitored in this example in red. A change ofelectric current was observed in response to each addition of glucose,ascorbic acid (AA) and acetaminophen (AP). However, after the additionof maltose, no electric current change greater than 5 nA/mMcm² wasobserved except the peaks caused by stirring. Accordingly, themaltose-blocking layer in this example effectively blocked maltose whilenot interrupting sensing of glucose.

Example 13.4 Electrode with Maltose-Blocking Layer (0.1 mM, 100 mV/sec)

Examples 13.1-13.3 were repeated except that the working electrode 103included a maltose-blocking layer that was prepared as in Example 12.4(using 0.1 mM mPD solution at scanning rate of 100 mV/sec). FIG. 25shows the electric current monitored in this example in green. A changeof electric current was observed in response to each addition ofglucose, ascorbic acid and acetaminophen. However, after the addition ofmaltose, no electric current change greater than 5 nA/mMcm² was observedexcept the peaks caused by stirring. The maltose-blocking layer in thisexample effectively blocked maltose while not interrupting sensing ofglucose.

Example 13.5 Electrode with Maltose-Blocking Layer (0.1 mM, 200 mV/sec)

Examples 13.1-13.3 were repeated except that the working electrode 103included a maltose-blocking layer that was prepared as in Example 12.5(using 0.1 mM mPD solution at scanning rate of 200 mV/sec). FIG. 25shows electric current monitored in this example in purple. A change ofelectric current was observed in response to each addition of glucose,ascorbic acid and acetaminophen. However, after the addition of maltose,no electric current change greater than 5 nA/mMcm² was observed exceptthe peaks caused by stirring. The maltose-blocking layer in this exampleeffectively blocked maltose while not interrupting sensing of glucose.

Example 13.6 Electrode with Maltose-Blocking Layer (0.3 mM, 10 mV/sec)

Examples 13.1-13.3 were repeated except that the working electrodeincluded maltose-blocking layer prepared in Example 12.6 (using 0.3 mMmPD solution at scanning rate of 10 mV/sec). FIG. 26 shows the electriccurrent monitored in this example in red. A change of electric currentwas observed in response to each addition of glucose, ascorbic acid andacetaminophen. However, after the addition of maltose, no electriccurrent change greater than 5 nA/mMcm² was observed except the peakscaused by stirring. The maltose-blocking layer in this exampleeffectively blocked maltose while not interrupting sensing of glucose.

Example 13.7 Electrode with Maltose-Blocking Layer (0.3 mM, 100 mV/sec)

Examples 13.1-13.3 were repeated except that the working electrodeincluded maltose-blocking layer prepared in Example 12.7 (using 0.3 mMmPD solution at scanning rate of 100 mV/sec). FIG. 26 shows the electriccurrent monitored in this example in green. A change of electric currentwas observed in response to each addition of glucose, ascorbic acid andacetaminophen. However, after the addition of maltose, no electriccurrent change greater than 5 nA/mMcm² was observed except the peakscaused by stirring. The maltose-blocking layer in this exampleeffectively blocked maltose while not interrupting sensing of glucose.

Example 13.8 Electrode with Maltose-Blocking Layer (0.3 mM, 200 mV/sec)

Examples 13.1-13.3 were repeated except that the working electrodeincluded maltose-blocking layer prepared in Example 12.8 (using 0.3 mMmPD solution at scanning rate of 200 mV/sec). FIG. 26 shows the electriccurrent monitored in this example in purple. A change of electriccurrent was observed in response to each addition of glucose, ascorbicacid and acetaminophen. However, after the addition of maltose, noelectric current change greater than 5 nA/mMcm² was observed except thepeaks caused by stirring. The maltose-blocking layer in this exampleeffectively blocked maltose while not interrupting sensing of glucose.

Example 13.9 Electrode with Maltose-Blocking Layer (0.5 mM, 10 mV/sec)

Examples 13.1-13.3 were repeated except that the working electrodeincluded maltose-blocking layer prepared in Example 12.9 (using 0.5 mMmPD solution at scanning rate of 10 mV/sec). FIG. 27 shows the electriccurrent monitored in this example in red. A change of electric currentwas observed in response to each addition of glucose, ascorbic acid andacetaminophen. However, after the addition of maltose, no electriccurrent change greater than 5 nA/mMcm² was observed except the peakscaused by stirring. The maltose-blocking layer in this exampleeffectively blocked maltose while not interrupting sensing of glucose.

Example 13.10 Electrode with Maltose-Blocking Layer (0.5 mM, 100 mV/sec)

Examples 13.1-13.3 were repeated except that the working electrodeincluded maltose-blocking layer prepared in Example 12.9 (using 0.5 mMmPD solution at scanning rate of 100 mV/sec). FIG. 27 shows the electriccurrent monitored in this example in green. A change of electric currentwas observed in response to each addition of glucose, ascorbic acid andacetaminophen. However, after the addition of maltose, no electriccurrent change greater than 5 nA/mMcm² was observed except the peakscaused by stirring. The maltose-blocking layer in this exampleeffectively blocked maltose while not interrupting sensing of glucose.

Example 13.11 Electrode with Maltose-Blocking Layer (0.5 mM, 200 mV/sec)

Examples 13.1-13.3 were repeated except that the working electrodeincluded maltose-blocking layer prepared in Example 12.11 (using 0.5 mMmPD solution at scanning rate of 200 mV/sec). FIG. 27 shows the electriccurrent monitored in this example in purple. A change of electriccurrent was observed in response to each addition of glucose, ascorbicacid and acetaminophen. However, after the addition of maltose, noelectric current change greater than 5 nA/mMcm² was observed except thepeaks caused by stirring. The maltose-blocking layer in this exampleeffectively blocked maltose while not interrupting sensing of glucose.

Example 13.12 Electrode with Maltose-Blocking Layer (1.0 mM, 10 mV/sec)

Examples 13.1-13.3 were repeated except that the working electrodeincluded maltose-blocking layer that was prepared in Example 12.12(using 1.0 mM mPD solution at scanning rate of 10 mV/sec) and furthersubjected to electric shock as in Example 12.13. FIG. 28 shows theelectric current monitored in this example in red. A change of electriccurrent was observed in response to each addition of glucose, ascorbicacid and acetaminophen. However, after the addition of maltose, noelectric current change greater than 5 nA/mMcm² was observed except thepeaks caused by stirring. The maltose-blocking layer in this exampleeffectively blocked maltose while not interrupting sensing of glucose.

Example 13.13 Electrode with Maltose-Blocking Layer (1.0 mM, 100 mV/sec)

Examples 13.1-13.3 were repeated except that the working electrodeincluded maltose-blocking layer prepared in Example 12.14 (using 1.0 mMmPD solution at scanning rate of 100 mV/sec) and further with electricshock as in Example 12.13. FIG. 28 shows the electric current monitoredin this example in green. A change of electric current was observed inresponse to each addition of glucose, ascorbic acid and acetaminophen.However, after the addition of maltose, no electric current changegreater than 5 nA/mMcm² was observed except the peaks caused bystirring. The maltose-blocking layer in this example effectively blockedmaltose while not interrupting sensing of glucose.

Example 13.14 Electrode with Maltose-Blocking Layer (1.0 mM, 200 mV/sec)

Examples 13.1-13.3 were repeated except that the working electrodeincluded maltose-blocking layer prepared in Example 12.15 (using 1.0 mMmPD solution at scanning rate of 200 mV/sec) and further with electricshock. FIG. 28 shows the electric current monitored in this example inpurple. A change of electric current was observed in response to eachaddition of glucose, ascorbic acid and acetaminophen. However, after theaddition of maltose, no electric current change greater than 5 nA/mMcm²was observed except the peaks caused by stirring. The maltose-blockinglayer in this example effectively blocked maltose while not interruptingsensing of glucose.

Example 13.15 Electrode with Maltose-Blocking Layer (2.0 mM, 10 mV/sec)

Examples 13.1-13.3 were repeated except that the working electrodeincluded maltose-blocking layer that was prepared in Example 12.16(using 2.0 mM mPD solution at scanning rate of 10 mV/sec) and furtherwith electric shock as in Example 12.15. FIG. 29 shows the electriccurrent monitored in this example in red. A change of electric currentwas observed in response to each addition of glucose, ascorbic acid andacetaminophen. However, after the addition of maltose, no electriccurrent change greater than 5 nA/mMcm² was observed except the peakscaused by stirring. The maltose-blocking layer in this exampleeffectively blocked maltose while not interrupting sensing of glucose.

Example 13.16 Electrode with Maltose-Blocking Layer (2.0 mM, 100 mV/sec)

Examples 13.1-13.3 were repeated except that the working electrodeincluded maltose-blocking layer prepared in Example 12.17 (using 2.0 mMmPD solution at scanning rate of 100 mV/sec) and further with electricshock as in Example 12.15. FIG. 29 shows the electric current monitoredin this example in green. A change of electric current was observed inresponse to each addition of glucose, ascorbic acid and acetaminophen.However, after the addition of maltose, no electric current changegreater than 5 nA/mMcm² was observed except the peaks caused bystirring. The maltose-blocking layer in this example effectively blockedmaltose while not interrupting sensing of glucose.

Example 13.17 Electrode with Maltose-Blocking Layer (2.0 mM, 200 mV/sec)

Examples 13.1-13.3 were repeated except that the working electrodeincluded maltose-blocking layer prepared in Example 12.18 (using 2.0 mMmPD solution at scanning rate of 200 mV/sec) and further with electricshock as in Example 12.15. FIG. 29 shows the electric current monitoredin this example in purple. A change of electric current was observed inresponse to each addition of glucose, ascorbic acid and acetaminophen.However, after the addition of maltose, no electric current changegreater than 5 nA/mMcm² was observed except the peaks caused bystirring. The maltose-blocking layer in this example effectively blockedmaltose while not interrupting sensing of glucose.

Example 13.18 Electrode with Maltose-Blocking Layer (5.0 mM, 10 mV/sec)

Examples 13.1-13.3 were repeated except that the working electrodeincluded maltose-blocking layer that was prepared in Example 12.19(using 5.0 mM mPD solution at scanning rate of 10 mV/sec) and furtherwith electric shock as in Example 12.15. FIG. 30 shows the electriccurrent monitored in this example in red. A change of electric currentwas observed in response to each addition of glucose, ascorbic acid andacetaminophen. However, after the addition of maltose, no electriccurrent change greater than 5 nA/mMcm² was observed except the peakscaused by stirring. The maltose-blocking layer in this exampleeffectively blocked maltose while not interrupting sensing of glucose.

Example 13.19 Electrode with Maltose-Blocking Layer (5.0 mM, 100 mV/sec)

Examples 13.1-13.3 were repeated except that the working electrodeincluded maltose-blocking layer prepared in Example 12.20 (using 5.0 mMmPD solution at scanning rate of 100 mV/sec) and further with electricshock as in Example 12.15. FIG. 30 shows the electric current monitoredin this example in green. A change of electric current was observed inresponse to each addition of glucose, ascorbic acid and acetaminophen.However, after the addition of maltose, no electric current changegreater than 5 nA/mMcm² was observed except the peaks caused bystirring. The maltose-blocking layer in this example effectively blockedmaltose while not interrupting sensing of glucose.

Example 13.20 Electrode with Maltose-Blocking Layer (5.0 mM, 200 mV/sec)

Examples 13.1-13.3 were repeated except that the working electrodeincluded maltose-blocking layer prepared in Example 12.21 (using 5.0 mMmPD solution at scanning rate of 200 mV/sec) and further with electricshock as in Example 12.15. FIG. 30 shows the electric current monitoredin this example in purple. A change of electric current was observed inresponse to each addition of glucose, ascorbic acid and acetaminophen.However, after the addition of maltose, no electric current changegreater than 5 nA/mMcm² was observed except the peaks caused bystirring. The maltose-blocking layer in this example effectively blockedmaltose while not interrupting sensing of glucose.

Example 13.21

Electrode with Maltose-Blocking Layer (1.0 mM, 10 mV/sec)

Example 13.12 is repeated except that the poly-mPD layer prepared inExample 12.12 (using 1.0 mM mPD solution at scanning rate of 10 mV/sec)is not subjected to electric shock.

Example 13.22 Electrode with Maltose-Blocking Layer (1.0 mM, 100 mV/sec)

Example 13.12 is repeated except that the poly-mPD layer prepared inExample 12.14 (using 1.0 mM mPD solution at scanning rate of 100 mV/sec)is not subjected to electric shock.

Example 13.23 Electrode with Maltose-Blocking Layer (1.0 mM, 200 mV/sec)

Example 13.12 is repeated except that the poly-mPD layer prepared inExample 12.15 (using 1.0 mM mPD solution at scanning rate of 200 mV/sec)is not subjected to electric shock.

Example 13.24 Electrode with Maltose-Blocking Layer (2.0 mM, 10 mV/sec)

Example 13.12 is repeated except that the poly-mPD layer prepared inExample 12.16 (using 2.0 mM mPD solution at scanning rate of 10 mV/sec)is not subjected to electric shock. No change of electric current isobserved in response to each addition of glucose, which means thepoly-mPD layer effectively blocks glucose.

Example 13.25 Electrode with Maltose-Blocking Layer (2.0 mM, 100 mV/sec)

Example 13.12 is repeated except that the poly-mPD layer prepared inExample 12.17 (using 2.0 mM mPD solution at scanning rate of 100 mV/sec)is not subjected to electric shock. No change of electric current isobserved in response to each addition of glucose, which means thepoly-mPD layer effectively blocks glucose.

Example 13.26 Electrode with Maltose-Blocking Layer (2.0 mM, 200 mV/sec)

Example 13.12 is repeated except that the poly-mPD layer prepared inExample 12.18 (using 2.0 mM mPD solution at scanning rate of 200 mV/sec)is not subjected to electric shock. No change of electric current isobserved in response to each addition of glucose, which means thepoly-mPD layer effectively blocks glucose.

Example 13.27 Electrode with Maltose-Blocking Layer (5.0 mM, 10 mV/sec)

Example 13.12 is repeated except that the poly-mPD layer prepared inExample 12.19 (using 5.0 mM mPD solution at scanning rate of 10 mV/sec)is not subjected to electric shock. No change of electric current isobserved in response to each addition of glucose, which means thepoly-mPD layer effectively blocks glucose.

Example 13.28 Electrode with Maltose-Blocking Layer (5.0 mM, 100 mV/sec)

Example 13.12 is repeated except that the poly-mPD layer prepared inExample 12.20 (using 5.0 mM mPD solution at scanning rate of 100 mV/sec)is not subjected to electric shock. No change of electric current isobserved in response to each addition of glucose, which means thepoly-mPD layer effectively blocks glucose.

Example 13.29 Electrode with Maltose-Blocking Layer (5.0 mM, 200 mV/sec)

Example 13.12 is repeated except that the poly-mPD layer prepared inExample 12.21 (using 5.0 mM mPD solution at scanning rate of 200 mV/sec)is not subjected to electric shock. No change of electric current isobserved in response to each addition of glucose, which means thepoly-mPD layer effectively blocks glucose.

Alternative Electric Shock Example 14.1 Electric Shock in two Pulses

Example 12.13 is repeated except that two pulses with the pulse width of0.5 sec. with an interval of 0.5 sec.

Example 14.2 Electric Shock in Two Pulses

Example 14.1 is repeated except that each pulse is from +0.0 V to +2.0V.

Example 14.3 Electric Shock in Multiple Pulses

Example 12.13 is repeated except that a series of 10 pulses with thepulse width of 0.1 sec. with an interval of 0.1 sec. between two pulses.

Example 14.4 Electric Shock in Multiple Pulses

Example 14.1 is repeated except that each pulse is from +0.0 V to +2.0V.

Example 14.5 Electric Shock in a Single Ramp

Example 12.13 is repeated except that the electric potential graduallyincreases from +0.0 V to +1.0 V. during the period of 1 sec.

Example 14.6 Electric Shock in Multiple Ramps

Example 14.5 is repeated except that the ramp electric potential isrepeated 5 times with an interval of 0.1 between two ramps.

Example 14.7 Electric Shock in a Single Ramp

Example 12.13 is repeated except that the electric potential graduallyincreases from +0.0 V to +2.0 V. during the period of 2 sec.

Example 14.8 Electric Shock in Multiple Ramps

Example 14.7 is repeated except that the ramp electric potential isrepeated 5 times with an interval of 0.1 between two ramps.

Conditioning Working Electrode Example 15.1 Preparing Glucose-SensingSystem in Serum

Example 10.2 was repeated to prepare an electrochemical cell for glucosesensing in serum. The working electrode 103 was one of the electrodes1607 (including platinum nanoporous layer 1609) prepared in Example 8.4and does not include an electrolyte ion-blocking layer.

Example 15.2 Conditioning Working Electrode (No Electrolyte Ion-BlockingLayer)

In the electrochemical cell prepared in Example 15.1, the bias voltageof 0.4 V was applied between the working electrode 103 and the referenceelectrode 106. Unlike in Example 10.3, immediately upon applying thebias voltage, electric current from the working electrode wascontinuously measured. FIG. 42A shows a profile of the electric currentprofile measured from the electrochemical cell, in which the workingelectrode 103 does not include an electrolyte ion-blocking layer.Referring to FIG. 42A, at 10,000 seconds (about 3 hours), 20,000 secondsand 30,000 seconds, the electric current still decreases at asignificant rate. FIG. 42B is an enlarged view of the profile of FIG.42A and shows that the glucose stock solution prepared as in Example 9.1was added well after the completion of conditioning of the workingelectrode.

Example 15.3 Preparing Working Electrode with PMMA ElectrolyteIon-Blocking Layer

PMMA purchased from Sigma-Aldrich (Product No. 445746) was dissolved indimethylformamide (DMF) to provide 2 wt % PMMA solution. Using amicro-syringe, 0.2 μL of the PMMA solution was dropped on the platinumnanoporous layer 1609 of one of the electrodes 1607 prepared in Example8.4. When the solvent dried off, a PMMA electrolyte ion-blocking layer505 was formed on the platinum nanoporous layer 1609.

Example 15.4 Preparing Glucose-Sensing System in Serum

Example 10.2 was repeated for preparing an electrochemical cell forglucose sensing in serum except that the working electrode with PMMAelectrolyte ion-blocking layer prepared in Example 15.1 was used asworking electrode 103.

Example 15.5 Conditioning Working Electrode

In the electrochemical cell prepared in Example 15.4, the bias voltageof 0.4 V was applied between the working electrode 103 and the referenceelectrode 106. Immediately upon applying the bias voltage, electriccurrent from the working electrode was continuously measured. FIG. 43shows a profile of the electric current measured from theelectrochemical cell, in which the working electrode 103 includes anelectrolyte ion-blocking layer. The glucose stock solution prepared asin Example 9.1 was added well after the completion of conditioning ofthe working electrode. The peaks in FIG. 43 represent stirring aftereach addition.

Example 15.6 Comparing Conditioning Time

FIG. 44 overlays the electric current profiles of FIG. 42 (Example 15.2)and FIG. 43 (Example 15.5). The electric current of Example 15.5(including an electrolyte ion-blocking layer) has settled and stabilizedin about 600 sec whereas the electric current of Example 15.2 (noelectrolyte ion-blocking layer) decreases at a significant rate in thesame time frame.

Example 15.7 Preparing Working Electrode with PHEMA Layer

PHEMA purchased from Sigma-Aldrich (Product No. 529265) was dissolved indimethylformamide (DMF) to provide 2 wt % PHEMA solution. Using amicro-syringe, 0.2 μL of the PHEMA solution was dropped on the platinumnanoporous layer 1609 of one of the electrodes 1607 prepared in Example8.4. When the solvent dried off, a PHEMA electrolyte ion-blocking layer505 was formed on the platinum nanoporous layer 1609.

Example 15.8 Preparing Working Electrode with PMMA-EG-PMMA Layer

PMMA-EG-PMMA purchased from Sigma-Aldrich (Product No. 463183) wasdissolved in dimethylformamide (DMF) to provide 2 wt % PMMA-EG-PMMAsolution. Using a micro-syringe, 0.2 μL of the PMMA-EG-PMMA solution wasdropped on the platinum nanoporous layer 1609 of one of the electrodes1607 prepared in Example 8.4. When the solvent dried off, a PMMA-EG-PMMAelectrolyte ion-blocking layer 505 was formed on the platinum nanoporouslayer 1609.

Example 15.8 Preparing Glucose-Sensing Systems and Conditioning in Serum

Electrochemical cells for glucose sensing in serum were prepared byrepeating Example 15.4 except that the working electrodes prepared inExamples 15.7 and 15.8 were used as working electrode 103. Further,Example 15.5 was repeated for the prepared electrochemical cells.

Making CGM Subcutaneous Electrode Unit Example 16.1 Forming ConductiveLayer on Base

A polyimide film with the thickness of 150 μm was used as a basesubstrate 503. A silver layer 1603 was printed on the polyimide film toprovide about 20 μm thickness of silver conductive elements 110C, 110Wand 110R in the shapes as illustrated in FIG. 35. Subsequently, aconductive carbon layer 1605 was printed on the silver conductiveelements 110C and 110W in the thickness of about 20 μm. No carbon layerwas formed on the silver layer conductive element 110R.

Example 16.2 Placing Insulation Layer and Cutting

A polyimide film with the thickness of 50 μm was used as an insulationlayer 707. The polyimide film was cut in a size to cover theintermediate product of FIG. 35 while exposing the terminal portion 705.The polyimide film was punctured to provide three openings for exposingareas for working, reference and counter electrodes. Subsequently, thepre-cut polyimide was placed over the intermediate product of FIG. 35such that the adhesive layer contacts the polyimide base 503 forproviding the intermediate product of FIG. 36. Subsequently, thepolyimide base 503 and polyimide insulation layer 707 outside theconductive elements were cut to provide an intermediate product of FIG.37.

Example 16.3 Forming Clustered Nanoporous Layer

The cluster colloid obtained in Example 5.1 was diluted to 60 mg/ml withpurified water. Using a micro-syringe, 0.2 μL of the diluted clustercolloid was dropped on the carbon layer 1605 exposed through one openingfor the working electrode 501 of the intermediate product prepared inExample 16.2. The cluster colloid dropped on the carbon layer 1605 wasdried to provide the clustered nanoporous layer 117, resulting in anintermediate product of FIG. 38A.

Example 16.4 Forming Electrolyte Ion-Blocking Layer

PMMA purchased from Sigma-Aldrich (Product No. 445746) was dissolved indimethylformamide (DMF) to provide 2 wt % PMMA solution. Using amicro-syringe, 0.2 μL of the PMMA solution was dropped on the nanoporouslayer 117 of the intermediate product prepared in Example 16.3. When thesolvent dried off, the PMMA electrolyte ion-blocking layer 505 wasformed on the nanoporous layer 117.

Example 16.5 Forming Biocompatibility Layer

A biocompatibility layer (pHEMA) is formed on the electrolyteion-blocking layer 505 as illustrated in FIG. 38B, resulting in anon-enzymatic CGM electrode unit of FIG. 33.

Example 16.6 Forming Biocompatibility Layer

pHEMA purchased from Sigma-Aldrich (Product No. 192066) was dissolved indimethylsulfoxide (DMSO) to provide 0.5 wt % pHEMA solution. Using amicro-syringe, 1.0 μL of the pHEMA solution was dropped on theelectrolyte ion-blocking layer 505 of the intermediate product preparedin Example 16.4. When the solvent dried off, the pHEMA biocompatibilitylayer 507 was formed as illustrated in FIG. 38B, resulting in anon-enzymatic CGM electrode unit 701 of FIG. 33.

CGM Animal Testing Example 17.1 Preparation for CGM Animal Testing

The non-enzymatic CGM electrode unit prepared in Example 16.6 wassubcutaneously inserted into a rat's body such that the electrodes 103,105 and 106 contact interstitial fluid of the rat. The CGM electrodeunit 701 was connected to a UXN potentiostat developed by UXN Co., Ltd.(Applicant of the present application) and Seoul National UniversityHospital. FIG. 45A is a photograph of the UXN potentiostat. FIG. 45B isa photograph showing that the CGM electrode unit 701 is connected to theUXN potentiostat of FIG. 45A. FIG. 45C is a photograph showing that theUXN potentiostat with its case. The UXN potentiostat includes a wirelessmodule for wirelessly communicating with a computer and can bewirelessly controlled by the computer. A glucose solution was preparedfor injecting into the rat's vein to cause changes of the glucose levelin the rat's blood and interstitial fluid.

Example 17.2 Continuous Monitoring of Rat's Glucose Level

Subcutaneous insertion of the CGM electrode unit 701 was maintained for5 consecutive days. On the first day, the glucose solution was injectedto the rat twice. On the following days, the glucose solution wasinjected once a day. The UXN potentiostat measured the electric currentfrom the CGM electrode unit 701 over a time span of about 1.5 hoursafter the (first) injection each day. Also, every 2-5 minutes during thetime span of about 1.5 hours, a small amount of the rat's blood wastaken from its tail and applied to a test strip for Roche Accu Chek®blood glucose meter, which provided a glucose concentration in theblood.

Example 17.3 Plotting CGM Measurements and Blood Glucose of Rat

FIG. 46 shows the electric current from the CGM electrode module in bluethat was measured by the UXN potentiostat in Example 17.2. The red dotsof FIG. 46 represent the blood glucose concentrations obtained from theRoche Accu Chek® blood glucose meter. Given that there is a time lag ofabout 10 minutes between the glucose level in interstitial fluid and theglucose level in blood, the data were calibrated by shifting bluesignals shifted relative to the red dots in time. It is understood thatsharp peaks in the blue signals are primarily from the rat's physicalmovements during the measurements. Based on the graph of FIG. 45, thereappears to be a strong correlation between the blood glucoseconcentrations using the Roche Accu Chek® blood glucose meter and theCGM monitoring using the non-enzymatic CGM electrode unit 701 preparedin Example 16.6.

Example 17.4 Clarke Error Grid Analysis

FIG. 47 is Clarke Error Grid for the non-enzymatic CGM electrode unit701 prepared in Example 16.6 based on the measurements presented in thegraph of FIG. 46. The reference sensor for this Clarke Error GridAnalysis is the Roche Accu-Chek® blood glucose meter. The grid has fiveregions. Region A includes values within 20% of the reference sensor;Region B includes values that are outside of Region A's 20% but wouldnot lead to inappropriate treatment; Region C includes values that arepotentially leading to unnecessary treatment; Region D includes valuesindicating a potentially dangerous failure to detect hypoglycemia orhyperglycemia; and Region E includes values that would confuse treatmentof hypoglycemia for hyperglycemia and vice versa. As summarized in thetable below the grid, the analysis shows that over 91% of the dots werein Region A and Region B.

Combination Of Features

This disclosure provide a lot of discussions and information about manyfeatures relating to nanoporous structures and/or glucose sensingtechnologies. It is the intention of this disclosure to provide as manydevices, systems and methods relating to those features. Two or morefeatures disclosed above may be combined together to form a device,system or method to the extent they are combinable even if a particularcombination is not presented in the present disclosure. Also, it is theintention of this disclosure to pursue claims directed to many of thosefeatures disclosed herein. Some of those features are presented in theform of claims in following section. Many claims are presented independent form by referring to one or more other claims. Applicant notesthat some claims referring to multiple claims may encompass acombination of features that are in conflict with one another(hereinafter “improper combination”). However, Applicant recognizes thatsuch claims may still encompass one or more combinations of featuresthat do not have any conflicts with one another (hereinafter “propercombination”). By presenting claims that may encompass both proper andimproper combinations, Applicant confirms its or inventor's possessionof the proper combinations and intends to provide specific support forthe proper combinations for later claiming of those proper combinations.

What is claimed is:
 1. A method of making a nanoporous layer, the methodcomprising: dispensing on a substrate a colloid composition comprising aliquid and a number of nanoparticle clusters, wherein each nanoparticlecluster comprises a number of nanoparticles; and subjecting thedispensed colloid composition to drying to cause nanoparticle clustersto get close to one another and settle over the substrate as the liquiddries off, which deposits irregularly shaped bodies comprisingnanoparticles to form a nanoporous layer over the substrate, wherein theirregularly shaped bodies are connected to one another to provide athree-dimensional interconnected network of irregularly shaped bodies inthe nanoporous layer.
 2. The method of claim 1, wherein adjacentnanoparticle clusters abut one another as the liquid dries off duringdrying, which forms abutments between at least some adjacent ones of theirregularly shaped bodies while forming unoccupied spaces that are notoccupied by the irregularly shaped bodies between the at least someadjacent ones of the irregularly shaped bodies.
 3. The method of claim2, wherein the abutments between the at least some adjacent ones of theirregularly shaped bodies connect the at least some adjacent ones of theirregularly shaped bodies, which continues to other ones of theirregularly shaped bodies to form the three-dimensional interconnectednetwork of irregularly shaped bodies.
 4. The method of claim 2, whereinthe unoccupied spaces are irregularly shaped and connect with otherunoccupied spaces formed between other ones of the irregularly shapedbodies, wherein connections between the unoccupied spaces form athree-dimensional interconnected network of irregularly shaped spaces,wherein the three-dimensional interconnected network of irregularlyshaped spaces and the three-dimensional interconnected network ofirregularly shaped bodies are complementary to form the nanoporouslayer.
 5. The method of claim 1, wherein the three-dimensionalinterconnected network of irregularly shaped bodies comprises numerousnanoparticles from the nanoparticle clusters of the colloid composition,wherein, inside the three-dimensional interconnected network ofirregularly shaped bodies, at least part of the nanoparticles areadjacent to each other without an intervening nanoparticle therebetweenand apart from each other with interparticular nanopores therebetween.6. The method of claim 5, wherein the interparticular nanopores aredistributed generally throughout in the three-dimensional interconnectednetwork of irregularly shaped bodies.
 7. The method of claim 5, whereinat least part of the interparticular nanopores inside thethree-dimensional interconnected network of irregularly shaped bodieshas an interparticular gap distance between two immediately adjacentnanoparticles, wherein the interparticular gap distance is within arange between about 0.5 nm and about 3 nm.
 8. The method of claim 1,wherein the nanoporous layer comprises the three-dimensionalinterconnected network of irregularly shaped bodies and furthercomprises a three-dimensional interconnected network of irregularlyshaped spaces that are not occupied by the three-dimensionalinterconnected network of irregularly shaped bodies.
 9. The method ofclaim 8, wherein at least part of the irregularly shaped spaces of thethree-dimensional interconnected network of irregularly shaped spacescomprises gaps in a size ranging between about 100 nm and about 500 nm.10. The method of claim 1, wherein at least part of the nanoparticles inthe irregularly shaped bodies is in a generally oval or spherical shapewith a length ranging between about 2 nm and about 5 nm.
 11. The methodof claim 1, wherein the colloid composition is substantially free of asurfactant, and accordingly the nanoporous layer deposited over thesubstrate is substantially free of a surfactant.
 12. The method of claim1, wherein the substrate comprises a surface that is electricallyconductive or semiconductive, wherein the three-dimensionalinterconnected network in the nanoporous layer is electricallyconductive and electrically connected to the surface.
 13. The method ofclaim 1, wherein the substrate comprises an electrically conductivemetal layer and an electrically conductive carbon layer formed over theelectrically conductive metal layer, wherein the nanoporous layer isformed on the electrically conductive carbon layer.
 14. The method ofclaim 1, wherein the colloid composition is dispensed in a predeterminedamount to form the nanoporous layer having roughness factor betweenabout 100 and about
 2500. 15. The method of claim 1, wherein the methodfurther comprises preparing the colloid composition, which comprises:providing a liquid composition comprising a surfactant and ions, whereinthe surfactant is in a reverse micelle phase comprising a plurality ofhydrophilic spaces; adding a reducing agent to the liquid composition tocause reduction of at least part of the ions to form nanoparticles,which provides a first colloid, wherein at least some of the hydrophilicspaces enclose at least one of the nanoparticles, wherein no electricpotential is applied for the reduction of at least part of the ions; andremoving the surfactant from the first colloid, which causes at leastpart of the nanoparticles to get together and form the nanoparticleclusters, each of which comprises a number of nanoparticles from thefirst colloid.
 16. The method of claim 15, wherein the nanoporous layeris substantially free of the surfactant such that the surfactant is inan amount smaller than 0.5 parts by weight with reference to 100 partsby weight of the nanoparticles contained in the nanoporous layer. 17.The method of claim 15, further comprising, subsequent to removing thesurfactant and prior to dispensing, storing the colloid composition in acontainer for a period longer than a week.
 18. The method of claim 15,further comprising, subsequent to removing the surfactant and prior todispensing, adding a solvent to the nanoparticle clusters to adjust aconcentration of the nanoparticles therein, which provides the colloidcomposition for dispensing.
 19. The method of claim 15, wherein somemolecules of the surfactant are bound to nanoparticles in the firstcolloid, wherein removing the surfactant further comprises adding anacid or base to the first colloid to separate molecules of thesurfactant from at least part of the nanoparticles in the first colloid.20. The method of claim 15, wherein removing the surfactant comprises:centrifuging the first colloid; collecting a bottom portion from acentrifuged composition; adding a solvent to the collected bottomportion; and repeating a sequence of centrifuging the collected bottomportion with added solvent, collecting a bottom portion from itscentrifuged composition, and adding the solvent thereto.